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Kinetic Analysis of the Contribution of Base Flipping to the Substrate Specificity and Catalytic Activity of Human Alkyl...

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KINETIC ANALYSIS OF THE CONTRIBU TION OF BASE FLIPPING TO THE SUBSTRATE SPECIFICITY AND CA TALYTIC ACTIVITY OF HUMAN ALKYLADENINE DNA GLYCOSYLASE By AARTHY C. VALLUR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Aarthy C. Vallur

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This work is dedicated to my family whose unshakable be lief in me made me aspire.

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iv ACKNOWLEDGMENTS My gratitude to my parents exceeds expr ession. They always instilled in me the will and desire to achieve, aspire and hope in my abilities. I am indebted to my husband for being an enormous source of support a nd encouragement through the trying years of graduate school. I would also like to acknowledge the ment orship of Dr. Linda Bloom, for the countless lessons I learnt from her and the very rewarding year s I spent in her lab. Beyond that, acknowledgements will not be comp lete without recalling the inspiration I derived from my excellent uncle and aunts, all my teachers through school and college and my dear friends, notably, Bala ji Krishnaprasad of Gainesville.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT......................................................................................................................x ii CHAPTER 1 BACKGROUND AND SIGNIFICANCE....................................................................1 The Problem of Chemical Reactivity of Bases.............................................................1 Base Excision Repair--All Roads Lead to DNA..........................................................3 Patch Size in Base Excision Repair..............................................................................6 2 HUMAN ALKYLADENINE DNA GLYCOSYLASETHE MASTER FLIPPER.......................................................................................................................9 AAG Knockout Mice and Implications for Repair.....................................................11 Diversity in Substrate Choice--Uniqueness of AAG..................................................14 Crystal Structure of AAG and Implications For Catalysis.........................................15 Relevance of Studying Flipping in AAG....................................................................20 Role of Tyr-162 in Flipping................................................................................20 Locating Substrates in DNA --Needle in a Haystack...........................................22 3 EXPERIMENTAL PROCEDURES...........................................................................24 Cloning and Expression of Huma n Alkyladenine DNA Glycosylase 79 (AAG 79) and Its Mutants....................................................................................24 Subcloning of hAAG 79E125Q into pET-15b Vector............................................24 Purification of Human Al kyladenine DNA Glycosylase 79 and Mutants...............26 Synthesis and Purificatio n of Oligonucelotides..........................................................28 Radio-Labeling of Substrates and Annealing to Complement...................................29 Single Turnover Excision Assa y For Glycosylase Activity.......................................30 Multiple Turnover Assays for Glycosylase Activity..................................................32 Electrophoretic Mobility Shift Assa y (EMSA) for AAG binding activity.................32 Melting Temperature (Tm) Meas urements for Duplex DNA.....................................33

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vi Fluorescence Assay for Ethenoade nine-AAG Binding and Excision........................34 Stopped-Flow Fluorescence to Observe Flipping of A by AAG..............................35 4 EFFECTS OF HYDROGEN BONDING WITHIN A DAMAGED BASE PAIR ON THE ACTIVITY OF WI LD-TYPE AND DNA-INTERCALATING MUTANTS OF HUMAN ALKYLADE NINE DNA GLYCOSYLASE...................37 DNA Substrates and Sequences..................................................................................39 Mutations to Tyr-162 and Projected Consequences...................................................40 Base Excision and DNA Binding Activities of the Y162S Mutant............................41 Activity of the Y162F Mutant....................................................................................43 Base Excision by the Y162F Mutant...................................................................43 DNA Binding Ability of Y162F Mutant.............................................................48 Implications of Flipping in the Catalytic Efficiency of AAG....................................51 A Two-Step Selection Model for AAG Activity........................................................54 5 ACTIVITY OF HUMAN ALKYLA DENINE DNA GLYCOSYLASE IS SENSITIVE TO THE LOCAL SEQUEN CE CONTEXT OF THE DAMAGED BASE..........................................................................................................................5 6 DNA Substrates indicating Base Stacking and Hydrogen Bonding Partners to Hx...........................................................................................................................57 Base Stacking and Hydrogen Bonding Effects on Hx................................................59 Effects of Hydrogen Bonding Partners on Hx Excision in the Strong and Weak Base Stacking Context...........................................................................60 Effects of Base Stacking Partners on Binding to Hx Substrates by AAG...........63 Effects of Hydrogen Bonding Partners on Binding to Hx in the Strong and Weak Base Stacking Context...........................................................................64 Base Stacking and Hydrogen Bonding Effects on eA ................................................66 Effects of Base Stacking and Hydrogen Bonding Partners on A Excision by AAG............................................................................................................66 Effects of Base Stacking and Hydr ogen Bonding Partners on Binding to A Substrates by AAG.....................................................................................68 Melting Temperatures of Hx and A Substrates.........................................................68 Sequence Context Effects and Implications for AAG Activity..................................70 6 ACTIVITY AND STABILITY OF AAG DURING ASSAYS.................................75 Loss of AAG Activity Can Contri bute to Reduced Catalysis....................................76 Multiple Turnover of Hx Is Dependent on the Base Pairing Partner.........................81 Multiple and Single Turnover of A Present Different Pictures................................84 Optimization of Assay Conditions for Maximum AAG Activity..............................86 Conclusions about Activity and Stability of AAG During Assays.............................89

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vii 7 FLUORESCENCE ASSAYS TO OB SERVE BINDING AND EXCISION OF ETHENOADENINE BY AAG IN REAL TIME.......................................................91 Fluorescence of A During Binding by AAG............................................................92 Blueshift of A Emission upon Binding by E125Q....................................................94 Fluorescence of A upon Excision of A by AAG.....................................................96 Stopped-Flow Analysis of A Flipping by AAG........................................................98 Contribution of Fluorescence Experi ments to Understanding of AAG....................100 8 DISCUSSION AND FUTURE DIRECTIONS........................................................102 The Night-Watchman Model for AAG Activity......................................................103 Tyr-162 and the Flipping Equilibrium..............................................................104 Stability of Hx and the Flipping Equilibrium....................................................106 Not All Bases are Born Equal...........................................................................108 Future Directions......................................................................................................110 Completing the Need for a Real Ti me Assay to Measure Flipping..................110 Continuing Research on Sequence Context Effects..........................................111 Possible Strategy to Explain A Excision.........................................................111 Coordination of the Activity of AAG to Other Downstream Steps in BER.....113 LIST OF REFERENCES.................................................................................................115 BIOGRAPHICAL SKETCH...........................................................................................122

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viii LIST OF TABLES Table page 4-1 Sequences of DNA substrates and positions of damaged base pairs.......................40 4-2 Observed excision rates and relativ e acitivities of AAG and Y162F mutant..........47 5.1 Melting temperatures of Hx and A substrates and corresponding single turnover excision rates............................................................................................................70 6.1 Comparison of pre-incubation of 160nM AAG at 37 C with product formed and rates of product formation with 5nM THxA T........................................................78 6.2 Comparison of pre-incubation of 40nM AAG at 37 C with the product formed and rate of product formation with 5nM THxA T...................................................81 6-3 Observed rates of multiple to single tu rnover titration assays of AAG with 5nM A T.........................................................................................................................86 6.4 Comparison of the effects of pH and salt on AAG activity and stability with 5nM GHxC T...................................................................................................................88

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ix LIST OF FIGURES Figure page 1-1 Structures of damaged base s, commonly encountered in DNA.................................2 1-2 Mechanism of action of mono-a nd bi-functional DNA glycosylases........................5 1-3 Base excision and repa ir by different pathways.........................................................7 2-1 Basal promoter region of the AAG gene..................................................................11 2-2 Bases found to be in vitro substrates for AAG.........................................................15 2-3 Residues that intercalate into the mi nor groove when AAG flips the pyrrolidine abasic analog............................................................................................................17 2-4 Crystal structure of AAG bound to a pyrro lidine abasic nucleotide........................18 2-5 Crystal structure of E125Q bound to A..................................................................20 3-1 The map showing the multiple cloning site and the other features of the pET-15b vector........................................................................................................................2 5 3-2 A schematic of the excision assay and a sample gel for resolution of products from substrates.........................................................................................................32 3-3 Fluorescent properties of 100nM A when in double stranded DNA and after excision by 400nM AAG at 37 C for 60 minutes....................................................35 4-1 Chemical structures of hypoxanthine and 1, N6-ethenoadenine paired with thymine and difluorotoluene....................................................................................39 4-2 Projected differences in intercalat ion ability of Ser-162 and Phe-162 when compared to the wild type residue, Tyr-162, based on the crystal structure of AAG bound to A.....................................................................................................41 4-3 Electrophoretic mobility sh ift assays to measure the affinity of the Y162S mutant for DNA containing an AT or a HxT base pair...................................................42 4-4 Plots of time courses for Hx excision by wt AAG 79 and Y162F .........................45 4-5 Plots of time courses for A excision by wt AAG 79 and Y162F..........................46

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x 4-6 Control glycosylase assay to show that base pairing with F does not make A or G excisable by AAG....................................................................................................48 4-7 Binding of wt AAG 79 and the Y162F mutant to DNA containing HxT and HxF base pairs.........................................................................................................50 4-8 Binding of AAG and the Y162F mutant to DNA containing AT and AF base pairs.......................................................................................................................... 51 5-1 Chemical structures of Hx and A base paired to thymin e, diflorotoluene and cytosine.....................................................................................................................58 5-2 Single turnover excision of Hx opposite T with T-A and G-C base stacking partners.....................................................................................................................60 5-3 Single turnover excision of Hx with T-A and G-C base stack ing partners opposite non-hydrogen bonding base pairing partner, F........................................................62 5-4 Single turnover excision of Hx with T-A and G-C base stack ing partners opposite WatsonCrick hydrogen bonding partner, C ..........................................................63 5-5 Electrophoretic mobility shift assa ys to measure binding of AAG to DNA containing Hx in diffe rent sequence contexts .........................................................65 5-6 Single turnover excision of A with G-C stacking partners.....................................67 5-7 Single turnover excision of A opposite C with G-C base stacking partners..........67 5-8 Binding of E125Q to A substrates with G-C stacking partner s. .............................68 6-1 AAG death assay under si ngle turnover conditions.................................................77 6-2 AAG death assay under singl e turnover conditions.................................................80 6-3 Multiple turnover of Hx T and Hx F with G-C stacking partners..........................82 6-4 Multiple turnover of Hx T and Hx F with TG stacking partners............................83 6-5 Multiple to single turnover titration of AAG with A T.........................................85 6-6 Effect of pH and salt on excision of Hx T...............................................................88 6-7 Effect of BSA on AAG stability and activity...........................................................89 7-1 The 500 second time-based fluorescence of A when bound by increasing concentrations of E125Q .........................................................................................93 7-2 Emission spectra of 100 nM A substrates, after adding 25 to 800 nM E125Q at room temperature.....................................................................................................94

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xi 7-3 Emission scans of increasing concentr ations of E125Q only when excited at 320 nm......................................................................................................................95 7-4 Emission spectra of 100 nM TEG with 100, 200 and 80 0nM Y162S....................96 7-5 Timebased increase in A fluorescence upon excision by 800nM AAG at 37 C .........................................................................................................................98 7-6 Stoppedflow analysis of A flipping by AAG.....................................................100 8-1 The Night-watchman model for AAG activity......................................................104 8-2 Compromised flipping du e to Tyr-162 mutation...................................................105 8-3 Effect of Hx stability on the Night-watchman model.............................................108 8-4 Effect of A excision on the Night-watchman model............................................109 8-5 A hypoxanthine zebularine base pair...................................................................111 8-6 Crystal structure of the AAGA complex showing the pr oximity of Met-169 to the etheno bridge of the flipped out A in the active site...................................113

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xii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy KINETIC ANALYSIS OF THE CONTRIBU TION OF BASE FLIPPING TO THE SUBSTRATE SPECIFICITY AND CA TALYTIC ACTIVITY OF HUMAN ALKYLADENINE DNA GLYCOYSLASE By Aarthy C. Vallur December 2004 Chair: Linda B. Bloom Major Department: Biochemi stry and Molecular Biology Human alkyladenine DNA glycosylase (AAG) removes a variety of alkylated and deaminated bases from double stranded DNA to initiate base excision repair of damaged adenines. The crystal structure of AAG shows th at the enzyme uses a characteristic base flipping mechanism and does so by using Tyr-162 to intercalate through the minor groove and occupy the space vacated by the flip ped out substrate. The purpose of this dissertation is to further unders tand the contribution of base f lipping to the specificity and efficiency of AAG. A Y162S mutant showed undetectable activity on hypoxanthine (Hx) and 1, N6ethenoadenine ( A) substrates. A Y162F mutant show ed 2-fold reduced activity on Hx but the activity of the mutant was rescue d by a DNA mutation, in which the thymine (T) opposite Hx was replaced by diflorotoluene (F) with which Hx cannot base pair. The

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xiii reduced activity of the Y162F mutant on A was not rescued by the same DNA mutation. Other changes were made to affect the stability of Hx in DNA in an attempt to understand the importance of flipping to AAG activity on Hx. When Hx T was placed between strong base stacking G-C partners, excision of Hx was reduced and required 4 times the AAG needed to saturate th e excision of the same amount of Hx T placed between weak base stacking T-A partners. When T was replaced by F, excision of Hx in both sequence contexts was enhanced, but the enhancement with the G-C stacking partners was greatest, up to 10-fold and enhancement with T-A stacking partner was modest. When T was replaced by C with wh ich Hx forms WatsonCrick bonds excision was poor and the stacking pa rtners did not matter. The above results went on to show th at AAG could use the Tyr-162 mediated flipping mechanism to specifically bind and us e the flipping mechanism as an important step in recognizing the substrate. Factors that affect the flipping equilibrium which is the first step in catalysis, like th e stability of the damaged base in DNA will also affect the capacity of the enzyme to remove the dama ged base, ultimately. The sequence context of the damaged base may be a determining f actor in its recogniti on by DNA glycosylases and hence in the initiation of its repair by the base excision repair pathway.

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1 CHAPTER 1 BACKGROUND AND SIGNIFICANCE The Problem of Chemical Reactivity of Bases The bases in DNA can undergo chemical m odifications which can alter the genetic code and cause disturbances in the helical structure of DNA. Like any unsaturated, heterocyclic compound, a base in DNA can fall prey to attacks by water, reactive oxygen species and many endogenous a nd environmental agents ( 1-6 ). These chemical changes account for around 20,000 damages/cell every day. Al most a quarter of these arise due to the hydrolysis of the N -glycosidic bond between the ba se and the deoxyribose sugar leading to abasic or apurin ic/apyrimidinic (AP) residue s in DNA. Purines are more susceptible to hydrolysis of the N -glycosidic bond at an in vivo rate of >4500 depurinations/ cell ( 3, 7 ) whereas depyrimidination by hydrolys is occurs at a rate of less than that of depurinations. Abasic sites are extremely mutagenic and cytotoxic since they can lead to replication blocks a nd DNA strand breaks if not repaired ( 8 ). Deamination is another consequence of hydrolysis in normal bases. Oxidation is a major source of damage to bases. DNA bases have electrophilic carbons that can undergo oxidation to form mu tagenic lesions, primary of which is 7,8dihydro-8-oxoguanine (8-oxoG) ( 6, 9 ). Pyrimidine glycols and formamidopyrimidines are also products of oxidative damage (Fi gure 1-1). Oxidative metabolism leads to the accumulation of free radicals and products of lipid peroxidation like aldehydes which can form etheno adducts with aden ine, guanine and cytosine ( 3 ). Alkylation damage is also

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2 brought about by S-adenosyl methionine, an abundant methyl donor in cells, which can react with the ring n itrogens of purines ( 3 ) (Figure 1-1). Figure 1-1. Structures of damaged bases, commonly encountered in DNA. Deamination, alkylation and oxidation are chemical changes that contri bute to damaged bases in DNA ( See text ) Other cellular agents that can react with bases include nitric oxide and its derivatives. Nitric oxide ( NO) is an important second messenger in the body and is involved in many functions including pathwa ys signaling neurotrans mission and arterial wall relaxation ( 10 ). Nitric oxide is highly reactiv e with oxygen and forms nitrous anhydride which can deaminate the primary amin o groups of cytosine, 5-methyl cytosine, adenine and guanine ( 11, 12 ). NO induced deamination of cytosine, 5-methyl cytosine and guanine can lead to GC AT transitions, which are the primary mutations seen with NO. of the three, guanine has the greatest susceptibility to NO induced deamination,

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3 which modifies dG to xanthine ( 13 ). NO induced deaminati on of dA to hypoxanthine, which can result in AT GC transitions. Spencer etal observed significant accumulation of xanthine and hypoxanthine as the predominant lesions and a significant number of single strand breaks in HBE-1 cells, a human bronchial cell line, when the cells were exposed to 1mM nitrite at times greater than 60 min ( 14 ). More in vivo studies with cultured muscle nervous tissue cel l lines, in which nitric oxide has important functions is needed to learn about the degree and prev alence nitric oxide-induced deamination. Base Excision RepairAll Roads Lead to DNA The base damages discussed above are sma ll chemical changes that do not disrupt the structure of DNA drastically. But the pr oblem of accumulating even small damages in a vast sea of base pairs that make up the ge nome posts many challenges to the integrity of the genome. Many pathways have evolved to repair damaged DNA and restore genomic integrity, of which the repair of small, chemically damaged bases falls on the base excision repair pathway (BER) ( 15-20 ). Initiation of BER is accomplished by a damagespecific DNA glycosylases, which recognize its substrate and then excise the C1-N glycosidic bond between the ba se and the sugar (Figure 12). This leaves behind an abasic residue in DNA, which is then proce ssed by an AP endonuclease to create a nick 5 to the abasic site. DNA glycosylases ar e thus very important in sustaining base excision repair and ensuring its success. Up to eight human proteins have been reported to have DNA glycosylase activity. The substrate specificity of glycosyl ases is wide, with some glycosylases being selective for one substrate only while others have a broader substrate range. Examples for glycosylases specific for one damaged base only are UDG (uracil) and hOGG1 (8-oxoguanine) while hAAG which can act on various adenine lesions is a good example for glycosylases that can act on a group of substrates ( 16 ). In

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4 addition to having C1-N glycosylase activity, so me glycosylases also have an additional AP lyase activity. These are called bifunctional glycosylases in contrast to the former, which are monofunctional glycosylases. Bifunctional glycosylases, also called the Class I AP endonucleases, utilize an enzyme amino group as a nucleophile to form a Schiffbase intermediate with the abasic site which then undergoes either a or a elimination reaction to leave a nick. elimination leaves an unsaturated aldehyde with the 3phosphate and when followed by -elimination (Figure 1-2), leaves just a 3phosphate residue which cannot be extended by a polymerase ( 21 ). These 3termini can be acted on by a Class II AP endonuclease, which processes the 3phosphate residue to leave a 3OH residue which can then be extended by a polymerase. Repair is then completed when a DNA polymerase replaces the nucle otide and a DNA ligase seals the 3-OH and 5phosphate termini. Some bifunctional glycosylases like the hOGG1 and the bacterial FPG also have deoxyribophosphatase (dRPase) activity which can hydrolyze the abasic sugarphosphate residue ( 22 ). The major Class II endonucleases in E. coli are Exo III and Endo IV, Exo III being responsible for 90% of the activity under normal physiological conditions ( 23, 24 ) while Endo IV takes over during periods of oxidative and nitrosative stress ( 25, 26 ). The mammalian homolog of Exo III is the human AP endonuclease I ( aka APEX, APEI, Ref I), which is responsible for 95% of AP endonuclease activity in humans ( 22, 27, 28 ). After the action of the Cl ass II endonuclease in hu mans, deoxyribosephosphate lyase (dRPlyase) activity and synthesis to fill the gap is carried out by polymerase (Pol ). After this, DNA ligase III, which interacts with Pol through the XRCC1 protein, seals the nick to restore the original sequen ce in DNA. It is signifi cant to note that Pol

PAGE 18

5 lacks the proofreading activity of replicative polymerases and is prone to a relatively high frequency of errors during incorporation, to the tune of 1 mism atched nucleotide per 3000-5000 residues ( 29 ). These errors occur despite an induced fit mechanism in the active site of Pol Two combined events ensure error free gap filling by Pol First, DNA ligase III discriminates against joining ends with a 3 mismatch thereby, allowing for excision of the wrong residue and repl acement of the mismatched residue ( 30 ). Second, just like replicative polymerases ac t as holoenzymes with separate subunits encoding varied functions, a distinct human 3 exonuclease can cleave the mismatched 3 residue and allow for replacement. Recently, a human homolog of the E. coli Pol III holoenzyme component, Dna Q exonuclease, ha s been identified, which can correct Pol errors during BER and hence can be a ma jor candidate for error free repair ( 31 ). Such a proofreading step will gua rantee that correction of endogenous DNA damage does not by itself contribute to a hi gh frequency of mutations. Figure 1-2. Mechanism of action of monoa nd bi-functional DNA glycosylases. The first 3 steps illustrate excision of the C1-N glycosyl bond by nucleophilic attack by activated water in monofunctional DNA glycosylases like UDG. Steps 48 illustrate the formation of a Schiff-base intermediate between a lysine residue and the sugar followed by elimination.( Figure adapted from Scharer, O.D and Jiricny, J Bioessays, 23:270-281 2001)

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6 Patch Size in Base Excision Repair An alternative BER pathway to the single nucleotide replacement synthesis carried out by Pol has been observed in eukaryotes. Th is pathway involves the replacement of around 4-19 nucleotides by DNA polymerase mediated strand displacement ( 32, 33 ) and has been seen to require FEN1 and PCNA ( 34 ). One possible need for this longpatch pathway could be the presence of termini which are resistant to the dRP lyase activity of Pol ( 35, 36 ). In this case, a more processive po lymerase may cause strand displacement and leave FEN1 to cleave the flap. The resu lting nick is sealed by DNA ligase I. In reconstitution assays it wa s shown that, in the presence of PCNA and FEN1, Pol can also carry out longpatch repair ( 32, 37, 38 ), suggesting that the role of PCNA may not be to support processive repli cation, but activation of FEN 1 ( 37 ). There is speculation that the BER initiating DNA glycosylase ma y influence the choice of patch size. An added complexity is the observation that other factors may influence the patch size. Sung and Mosbaugh used a closed, circul ar plasmid with a sitedirected uracil or ethenocytosine ( C) in E coli and studied the re sulting patch size ( 39 ). In reconstitution experiments with various BER proteins, they discovered that DNA ligase mediated endjoining was the slowest step and also obser ved, short, long and very long patch repair. Increasing the ligase to polymerase ratio bias ed synthesis towards shortpatch repair. With long time periods, longer patches (> 20 nucleotides) were observed ( 39 ). The idea that the steps in BER are coordina ted has been given a lot of thought due to the complicated interplay of proteins and mounting evidence delineating the interactions between enzymes catalyzing succ essive steps. Interactions between APE1 and Pol ( 40 ), Pol and XRCC 1 ( 41, 42 ), Pol and ligase 1 ( 43 ) and XRCC1 and PARP ( 44 ) have been reported. The role of PARP in BER may be accidental but

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7 nevertheless important, as it is known to be a sensor for nicks in DNA and interacts with XRCC1 ( 44, 45 ). Moreover, PARP knockouts are highl y sensitive to methylnitrosourea, which leaves behind BER substrates in DNA ( 46 ). There is also evidence for some functional interactions between glycosylases and APE1. Th is stepwise coordination may be important in bringing substrates and enzymes together and al so protecting the cell from the harmful abasic sites that acc umulate as intermediates in BER. Figure 1-3. Base excision and repair by different pathways. Base excision, initiated by a damage specific DNA glycosylase can lead to resynthesis by either the short patch pathway, in which one nucleotid e is resynthesized or long patch pathway, in which anywhere from 420 nucleotides are resynthesized. Monoand bi-functional glycosylases leave be hind different 3 termini which are processed by AP endonucleases or Pol Abbreviations: XRCC1X-Ray Cross Complementation pr otein, PCNAProliferating Cell Nuclear Antigen, FEN1Flap-Endonuclease1, RF-CReplicating FactorC. Monofunctional DNA glycosylase AP endonuclease DNA pol SHORT-PATCH PATHWAY P DNA pol DNA ligase III/XRCC1 LONG-PATCH PATHWAY DNA pol or PCNA & RF-C FEN1 & PCNA DNA ligase I EnO O Bifunctional DNA glycosylase AP Lyase AP Endonuclease DNA Pol

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8 Though it is reasonable to assume that DNA glycosylases are localized to the nucleus where they function, it is also importa nt to address the problem of damage to mitochondrial DNA, especially given its pr oximity to the oxida tive phosphorylation system. It has been found that many euka ryotic DNA glycosylase mRNAs like those coding uracil DNA gl ycosylase (UDG) ( 47 ), human 8-oxoguanine DNA glycosylase (hOGG1) ( 48 ) and the human MutY homolog (MYH) ( 49 ) are alternatively spliced to encode nuclear and mitochondrial versions of the protein ( 50 ). Two additional DNA glycosylase activities have b een reported to be localized only to mitochondria, MtODE and MtGendo ( 51, 52 ). These act on oxidatively damaged bases. More research is needed to reconstitute the BER pathway in the m itochondria and identify other proteins and splice variants for other components of the pathway.

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9 CHAPTER 2 HUMAN ALKYLADENINE DNA GLYCOSYLASETHE MASTER FLIPPER Alkylating agents constitute one of the majo r offenders of the integrity of bases in DNA. Alkylated purines are mutagenic, espe cially susceptible functional groups in purines being the N7 of guanine and the N3 of adenine. Though commonly resulting from environmental and chemotherapeutic agen ts, alkylation damage can also result as a byproduct of cellula r activities. A unique suicidal enzyme, O6methylguanine methyl transferase directly reverses O6-methyl guanine lesions wh ich are highly prone to mispairing with thymine ( 53, 54 ). Most other alkylated ba ses are removed by specific DNA glycosylases and alkylated basespecific gl ycosylases have been cloned in almost all species studied, including bacteria, yeas t, mice and humans. With the discovery of UDG by Lindahl and coworkers in 1974 ( 55 ), came the awareness about the need for more glycosylases to play a role in base excision repair. Research since has identified E. coli glycosylases responsible for removing 3-methyladenine (3-meA) and other cytotoxic alkyl purines ( 56-58 ). In 1982, Goldthwait and cowo rkers identified two distinct glycosylases responsible for excising 3-meA, a 3-meA DNA glycosylase I constitutively expressed from the tag gene and a 3-meA glycosylase II which is encoded by the inducible alkA gene ( 59 ). The tag gene product was speci fic for 3-meA while the alkA product showed a broad substrate range including 3-meA, 3-meG and 7-meG. Subsequently, alkylpurine glycos ylase activities have been iden tified and purified from S. cerevisiae (MAG), mice (MPG) and humans (AAG). Alkyladenine DNA glycosylase (AAG) was identified as the hum an equivalent of E. coli AlkA and could complement tag

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10 and alkA deficient strains. It was indepe ndently characterized by three groups who alternatively named it as alkyl adenine DNA glycosylase (AAG) ( 60 ), methylpurine DNA glycosylase (MPG) ( 61 ) and alkyl-N-purine DNA glycosylase (ANPG) ( 62 ). It has been the only alkyl purine glycosylase discovere d in humans so far. In 1993, Vickers et al localized the human AAG gene between the globin gene cluster and the telomere of the short arm of chromosome 16 (chr16p) ( 63 ). Working with a colon adenocarcinoma cell line (HT29) and a human erytholeukemia cel l line (K562), they identified the gene as comprising five exons whose representation di ffered in the 5 end from the cloned cDNA reported earlier by Samson et al ( 60 ). Rafferty and coworkers, in 1994, screened a gt11 human placental cDNA library with a probe derived from the original sequence and identified two alternative cDNAs, which differed in seven N-terminal residues ( 64 ). They concluded that these were likely to be splice variants differing in exon usage and could be important in accounting for some properties such as cellular lo calization and binding properties of the protein. In a search for mo re information on the expression of AAG, O Connors group located the basal promoter within 80 bases of the start codon of exon 1 in HT-29, K562 and 3T3 cell lines ( 65 ). They also identified some putative transcription factor binding sites in the promoter region, including those for N-MYC, SP1, USF-1 and CBP. In super shift assays, N-MYC and SP1 were found to bind and super shift the promoter region (Figure 2-1). It was also reported that expression of the AAG gene was cellcycle dependent. The expression was seen to increase during G1, remain elevated during synthesis and then decrease to ba sal levels. This was consistent with DNA synthesis and the need for re pairing replicating DNA. Othe r BER enzymes like APE1 and

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11 Pol have been found to be induced upon accumulation of DNA damage. Whether AAG is also induced requires more extens ive in vivo studies to ascertain. Figure 2-1. Basal promoter region of the AAG gene. Putative binding sites for several transcription factors like USF-1, SP1, N-MYC and CBP were noticed (Adapted from Bouziane et al Mutation Research, 2000, 461:15-29) AAG knockout Mice and Implications for Repair Questions remained as to the precise biologi cal effects of methyladenines in the cell and the possible role of pr otection by AAG activity. Given the broad substrate range observed for the mouse 3-MeA gl ycosylase (MPG), homozygous AAG knockout mice were generated by two groups and examined fo r phenotypes that can make the role of AAG clearer. Engelward et al reported in 1997 that AAG was the major 3-MeA glycosylase in at least 4 diffe rent tissues, namely, liver, kidney, testes and lungs in these mice ( 66 ). Mouse embryonic fibroblasts that were deficient in AAG were more sensitive

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12 to Me-Lex, an alkylating agen t that generates 3-MeA specifi cally in DNA, than the wild type AAG producing fibroblasts. AAG was al so found to be the only glycosylase responsible for removal of Hx when tested in the testes, kidney and lung tissues. It was also the major activity responsible for excising A lesions, but it is interesting to note that though no other A glycosylase activity was noticed in testes and kidne y, there was a minor degree of A excision noticeable in the lungs of the AAG-/mice. Elder et al also published the generation and effects of the AAG-/genotype ( 67 ). They reported an increased frequency of MMSinduced mutations in the hprt gene of splenic Tlymphocytes afte r a single MMS dose of 150mg /kg. These mutations were mostly AT TA transversions (47%) with some GC TA transversions (27%). The mutation frequency was about 10-fold higher than the background. When analyzed, they found an accumulation of 3-MeA and methylated guanines in the hprt gene up to a 24hr period after MMS treatment, but found that O6-MeG and O4-MeT which are also formed at lower frequencies by MMS were e fficiently cleared in the knock out ( ko ) cells lacking MPG activity. A reason for the cytotoxicity of 3-MeA and 7-MeG are their labile glycosylic bonds depurinating at a higher ra te leading to abasic sites in the DNA. O6MeG and O4MeT are highly prone to mispairing and creating single base changes. O6MeG and O4MeT seem to be cleared by anothe r redundant activity, possibly by the nucleotide excision repair (NER) machin ery or another DNA glycosylase. Elder et al concluded that the importance of AAG may be more than the need to protect against 3MeA and 7MeG. It may be more importa nt to protect the genome from its other substrates, Hx, which is highly mutagenic and can cause AT GC transitions and A

PAGE 26

13 which is also promutagenic. Both these lesions have stable glycosidic bonds and may be the primary target for AAG. One remarkable feature of both these knoc kout mice was that both strains were active, viable, and fertile and did not show any other abnormalities, prompting the conclusion that either the spont aneous lesions that are the targets of AAG were not lethal when unrepaired or that othe r glycosylases/repair pathways can assist to handle these lesions. This is important because there is ev idence that, in yeast, NER can process some AAG substrates and can also act on some other methylated substrates like O6-MeG. It was recently reported that human AAG can in teract with the human RAD23 proteins, which are involved in recognition of dama ged bases in DNA in NER and that the interaction can functionally affect AAG bindi ng and excision activity on Hx containing DNA ( 68 ). Nevertheless, the AAG knockout mode l is the first glycosylase/BER homozygous knockout model available to better understand th e complex pathway. Knockouts of other downstream enzymes in BER have proved embr yonic lethal, possibly due to the general requirement of enzymes like Pol and APE1 for processing all products of glycosylase action. These enzymes may also have other roles to perform and any possible complementation in activity by the NER pathway may not be sufficient to make up for the loss of these enzymes in the knockout cells. Loss of individual DNA glycosylases, on the other hand, may be much easier to complement by other DNA glycosylases or pathways. Hence, hom ozygous knockout models of individual DNA glycosylases, like the AAG knockout mice discu ssed above, may be useful in generating heterozygous crosses to study th e effect of the BER machin ery and its interplay with other repair machineries.

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14 Diversity in Substrate Ch oiceUniqueness of AAG E. coli AlkA, mouse and human AAG have a ll been found capable of removing a diverse range of substrates unlike many other DNA glycosylases which are specific for one damaged base only. AAG was first identi fied as a 3-MeA glycosylase and hence named so ( 60, 62 ). But later, it was discovered to be able to remove 7-MeG, 8-oxoG, Hx and A bases in vitro (69-71) The AAG-/mice demonstrated that it was the major glycosylase responsible for th e removal of 3-MeA, Hx and A 8-oxoG may not be an AAG target since it was removed efficiently in the AAG ko cells probably by its own dedicated glycosylase, OGG1 ( 72 ). So being able to excise out at least 4 structurally diverse substrates in the cell raises the que stion of whether catalytic efficiency is compromised for substrate diversity. Met hylated adenines and guanines can arise spontaneously in vivo from the action of methyl donors like SAM. Methyl adenines are unstable and spontaneously depurinate at a hi gher rate than normal purines, leading to cytotoxic abasic sites in DNA, in addition to having the abilit y to block replication forks. Hx is deaminated adenine, formed by the action of nitric oxides and its derivatives in the cell. Nitric oxide is an important second messenger and is also released by activated macrophages. Hx is highly mutagenic. A on the other hand is formed by the action of lipid peroxidation products in the cell a nd the action of the common hepatocarcinogens, vinyl chloride and ethyl carbamate ( 62, 73, 74 ). In COS7 cells, 70% of A lesions led to mutations. So, the biological cost of compromised catalytic efficiency for substrate diversity can be immense, making AAG a unique ly gifted enzyme. How one active site can be tailormade to fit these diverse substrates is difficult to imagine. Extensive biochemical work done by our group show th at, AAG-mediated excision is sensitive to

PAGE 28

15 the base pairing partner of the damaged base, and the extent sensitivity varied with the identity of the damaged base ( 75, 76 ). Figure 2-2. Bases found to be in vitro substrates for AAG. 3-methyladenine, 7-methyl guanine, hypoxanthine and 1, N6-ethenoadenine were found to be excised by AAG in AAG-/cells and are italicized for emphasis. Crystal Structure of AAG and Implications for Catalysis The diverse substrate specificity of AAG a nd the fact that it is the only known alkylpurine excising glycosylas e makes it an interesting prot ein whose structure can not only tell us more about genera l glycosylase action but also about the complicated nature of substrate recognition used by AAG. Lau et al reported two crystal structures of AAG, 3-methyladenine N N N H N N H2CH3 + 7-methylguanine NH N N H N O NH2H3C + 3-methylguanine NH N N H N O N H2CH3 + hypoxanthine NH N N H N O 8-oxoguanine NH N N H H N O NH2O8-hydroxyguanine N H N N H N O NH2HO1, N 6 -ethenoadenine N N N H N N NH N N H N O N N N N H N O N Hethenoguanines

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16 one bound to a pyrrolidine abasic site analog ( 77 ) and another of the catalytically inactive AAG mutant bound to one of its substrates, ethenoadenine ( 78 ). Both have contributed enormously to the understanding of AAG a nd have provided a tremendous boost to research on AAG and its mechanism of action. The first reported structure was a 2.7 crystal structure of an enzymatically activ e fragment of AAG lack ing the first 79 amino acids in the N-terminal, bound to a pyrrolidi ne abasic nucleotide, which is a potent inhibitor of the glycosylase activity. This structure showed AAG as a single domain containing seven helices and eight strands. 3 4, in the core of the protein, protrude as a hairpin that inserts into the minor groove of the bound DNA, and displaces the target nucleotide, in this case the pyrrolid ine. The displacement is made possible by insertion of Tyr-162, so that it intercalates in the space occu pied by the nucleotide, which is then flipped out of the helix into the en zymes active site bindi ng pocket. This flipping mechanism was first identified in the cytosine methyl transferases ( 79, 80 ) and has also been consistently observed as the binding mechanism of choice for BER proteins like glycosylases ( 81-83 ) and endonucleases ( 84, 85 ). In the AAG-DNA complex the B-form DNA duplex is kinked away from the protein where Tyr-162 intercalates, by about 22 and is held by two clusters of basic residues that contact the DNA backbone on eith er side of the flipped out residue. The extent of the buried DNA surface, as measured with a 1.4 probe was 1034. This is similar to that observed with the UDG-DNA complex ( 81 ). The flipped out pyrrolidine is looped out of the helix by rotation of the P-O5 bond and the O3-P bond of the phosphodiester backbone on either side of the nucleotide. The intercalating Tyr162 pushes out the opposite T19 residue 1.5 into the major groove. No specific contacts

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17 between the opposing T19 and enzyme residues ar e visible in the flippedout nucleotideAAG structure. Met-164 and Tyr-165 located in the hairpin, fill the minor groove and push against the deoxyribose moieties of T19 and T9, thereby widening the minor groove by almost 2 3 to the flipped out pyrrolidi ne. This 3 distortion may help AAG to scan the DNA unidirectionally. Figure 2-3. Residues that in tercalate into the minor groove when AAG flips the pyrrolidine abasic analog ( Pyrr 7 ). Tyr-162 is fully intercalated between Guanine 6 and Thymine 8, while Thymine 19 ( T19 ) is pushed into the major groove. Met-164 and Tyr-165 widen the minor groove.

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18 Figure2-4. Crystal structure of AAG bound to a pyrroli dine abasic nucleotide. The enzyme binds by intercalating Tyr-162 in to the minor groove and flips out the abasic nucleotide into the active si te. Enzyme residues widen the minor groove (see text) (Adapted from Lau etal, Cell 95, 249258, 1998) The active site shows a central water mo lecule well positioned for a nucleophilic attack on the glycosyl bond of the flippedout nucleotide. It is linked by a hydrogen bonding network with the pyrrolidine nucleotid e and the side chains of Glu-125 and Arg182 and the main chain carbonyl of Val262. Glu-125 acts as a general base by deprotonating the water and th ereby activating it for attac k. Tyr-127 stabilizes Glu-125 by forming hydrogen bonds and may also stack against the base. The 2.1 crystal structure of AAG bound to A gives a clearer picture of how a substrate is bound in the active site. In this structure, Tyr-127 stacks against A while another active site tyrosine, Tyr-159 makes an edge to face stacking interaction with the A The flippedout base itself is rotated 85 about its glycosyl bond away from the double helix. It is remarkable that these active site residue s located in the buried core of AAG are conserved among the

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19 other putative alkylation repair glycosylases identified in Borrelia burgdorferi ( 86 ), Bacillus subtilis ( 87 ), Arabidopsis thaliana ( 88 ), and Mycobacterium tuberculosis ( 89 ). The other important family of DNA glycosylases, of which E. coli AlkA, S. cereviseae MAG and human OGG1 are members, binds D NA and flips out the target base through a structurally conserved helixha irpinhelix motif and use an aspartate as the catalytic residue, though there is no obvious sequence ho mology in this family of glycosylases. Whereas no AAG homologs have been noticed yet, in terms of its st ructural or sequence details, the conserved active site residues poi nt out that proteins with similar folds may employ similar catalytic mechanisms, especially given the divergence of the glycosylases that share these conserved resi dues, in contrast to the st ructural homology seen in the HhH family of glycosylases. The crystal structure also gives a hint of how AAG could achieve substrate specificity given its diverse substrate range ; yet exclude normal purines from its active site. Positively charged substrates like 3-MeA and 7-MeG are electron deficient and hence, they may be bound and excised by using the aromatic -electron stacking strategy in which, aromatic residues in the active site like Tyr127 stack favorably with the flipped out base, thereby stabilizing it for ex cision. This method of sandwiching electron deficient bases between aromatic residues is a hallmark of many met hylated base binding proteins such as AlkA and several mRNA binding proteins ( 82, 90 ). Add to it the intrinsic instability of the gl ycosylic bond in these bases, effective excision is more probable. But this same strategy may not work for neutral bases like Hx or A. The AAAG crystal structure shows that A is accommodated in the active site by a combination of aromatic stacking and hydrogen bonds betw een the main chain amide of His-136 and

PAGE 33

20 the N6 of the flipped out base. The stacking offe rs a lone pair of electrons which would not be possible if the base was ad enine. In the same manner, the O6 of Hx can accept a hydrogen bond from His-136, although guanine can do the same, its N2 amino group will clash with the side chain of Asn-169, there by restricting access to both normal purines but allowing both neutral substrates. This stra tegy for fitting the flipped nucleotide in the active site allows AAG to both screen for the right substrate and accommodate only the right substrate for excision, once flipped. Figure 2-5. Crystal structure of E125Q bound to A (left panel). The black A is flipped into the active site, where it stack s between Tyr-159, His-136.and Tyr-127. The active site is clearly shown in th e right panel. The orientation of the flipped out A relative to the catalytic residue and the water molecule is shown in a superposition of the active sites of E125Q/ A (green), wildtype AAG/ A (pink.) and wildtype AAG/ pyrrolidi ne abasic site (blue) (Adapted from Lau et al, Proc. Nat. Acad. Sci. USA 97-13573, 2000) Relevance of Studying Flipping in AAG Role of Tyr-162 in Flipping The flipping mechanism fac ilitated by insertion of Tyr-162 into the minor groove, is indicative of both how tight binding is achieved by AAG and how specific binding

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21 may be achieved. The limited DNA contacting surface seen in the crystal structure and the absence of any specific c ontacts between the opposite base and enzyme residues also increases speculation on how important Tyr-162 mediated intercalation is to the overall efficiency of substrate binding by AAG. In contrast, E. coli UDG, which flips out uracil from DNA, makes extensive contacts with the phosphodiester backbone of the DNA and involves a pinch-push-pull mechanism to extrude the uracil from the DNA into the active site ( 85 ). In this model, UDG, scans along the DNA using its leucine finger .When a U-G wobble base pair is detected by the leucine, the SerPro loops compress the phosphodiester backbone extensivel y, kinking the DNA to almost 45 thereby pinching the DNA to gain access to the uracil. This pinc hing gives the needed force for the leucine to intercalate into the minor groove thus, pus hing the uracil out of the helix where it is pulled into the active site by specific hydr ogen bonding interactions. The last two steps can happen in either order. It is clear how protei nDNA interactions other than leucine insertion can contribute to flipping uracil by UDG. But these interactions seem to be largely missing from the AAG crystal struct ure, in which we do not see extensive compression or kinking of the DNA to the exte nt seen with UDG. In addition, leucine mutations to alanine or gl ycine reduced UDG activity to 10% and 1% respectively ( 9193 ). In a similar attempt to te st the functional significance of Tyr-162 mutants and wildtype AAG were expressed in a S. cerevisiae strain lacking the endogenous yeast MAG1 and tested for resistance against the alkyla ting agent MMS. In contrast to the wt AAG, the Y162A mutant made yeast very sensit ive to MMS, pointing out that maybe the mutation may have rendered AAG unable to bi nd and hence excise alkylated bases. In contrast mutations to the two other residues, Met-164 and Tyr-165, which are also part of

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22 the -hairpin, and which are shown to assist flipping by destabiliz ing the base pair next to the flipped out base, did not confer MMS sensitivity to the yeast cells ( 78 ). Mutation of Tyr-162 to Ser reduced AAG activity to undetectab le levels, in kinetic studies done by us ( 75 ). So the importance of Tyr-162 to the proces s of flipping could be significant and in the absence of other f actors that could affect DNA bindi ng, must also contribute to the overall catalytic efficiency of the enzyme in ways more extensive than what is known from UDG. Locating Substrates In DNANeedle in a Haystack The crystal structure offers no plausible explanation of how the daunting task of identifying its substrates amidst millions of bases is accomplished in the vast genome with its complex hierarchy of structures. It does make sense though, to expect AAG to use Tyr-162 intercalation to scan the DNA or possible substrates. Since AAG substrates are not known to distort the backbone extensiv ely, a partial unstacking of the nucleotides may be needed for AAG to identify its substrate, as proposed for AlkA ( 77 ). Once a nucleotide is thus flipped, the dispropor tionate rigidity produced by Met164 and Tyr165 may help the protein progressively flip nucleotides in the 3 direction, thereby scanning the minor groove for substrates. Once a base is recognized, the interactions in the active site may include or exclude the base as a substrate for excision. This twostep recognition may also help AAG preserve its substrate diversity. An added complexity comes from the reports that, though AAG does no t seem to make any specific contacts with the opposite base, it is sensitive to th e opposite base in terms of binding and excision proficiency ( 75, 76 ). These findings further enha nce the need to understand the mechanism of AAG may achieve substrate diversity, given the different properties of the substrates it excises.

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23 A systematic analysis of the biochemistry of flipping and its effects will be very useful in understanding the mechanism of action of AAG and inching closer to the unsolvable riddle of how glycosylases find th eir substrates in the vast genome. This dissertation attempts to address the issue of the contribution of fli pping to the activity of AAG. A systematic, biochemical approach to analyzing the effects of mutating Tyr-162 on flipping efficiency and the contribution of local DNA sequence context to flipping efficiency was undertaken. The kinetic interp retation of the resulti ng observations reveals important biological implications about the co ntribution of flipping to the specificity and the catalytic efficiency of AAG ( 75 ). The conclusions drawn from this dissertation and the reagents developed to conduct the resear ch will be valuable in dictating further studies on AAG mechanism and action.

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24 CHAPTER 3 EXPERIMENTAL PROCEDURES Cloning and Expression of Human Alkyladenine DNA Glycosylase 79 (AAG 79) and Its Mutants A deletion mutant of AAG that is missi ng the first 79 amino acids from the Nterminus (AAG 79) was constructed using P CR, as already published ( 75, 76 ). Deletion of this unconserved N-terminal region has b een shown to have no effect on either base excision or DNA binding activities of the enzy me, but the truncated protein is more soluble at low ionic strength. All site-directed mutants were made in the coding sequence of this truncated gene using the Transf ormer Site-directed mutagenesis kit (BD Biosciences Clontech, Palo Alto, CA). The primers used to generate the desired mutations were also engineered to contain a si lent mutation that creat ed a restriction site to facilitate scr eening of clones. Subcloning of hAAG 79E125Q into pET-15b Vector The hAAG 79 constructs were cloned into p ET-14b vectors and were used to transform E. coli BL21 (DE3) cells. The hAAG 79 E125Q mutant was subcloned into a pET-15b vector due to its incompatibility with the pET-14b vector. The pET-15b vector has a multiple cloning site with a T7 promoter sequence and the lac operator sequence, which can allow for more controlled expressi on of the cloned gene and hence help get over the incompatibility problems seen w ith pET-14b. The E125Q gene sequence along with an upstream ribosome binding site was subcloned into the Xba1 and the BamH1 restriction sites (Figure 3-1). The vector was transfected into DH5cells and the plasmid

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25 was isolated from the cells. The sequence a nd alignment of the E125Q gene was checked by sequencing. All other AAG mutants we re cloned into pET-14b vectors. Figur 3-1. The map showing the multiple cloning site and the other features of the pET15b vector. The multiple cloning site a nd its components are shown in the sequence below the map. The restriction sites used to subclone the E125Q gene, the Xba1 and the BamH1 sites ar e highlighted in blue with arrows. The transformed cells were plated on 2xYT plates containing 100 g/mL ampicillin and grown for 16-20 hours at 37 C. A 10-mL aliquot of fresh 2xYTcontaining 100 g/mL ampicillin was inoculated with a single colony of transformed cells and grown at 37 C with vigorous shaking (200 rpm) to A600 of 0.5-0.8 and then stored at 4 C overnight. The

PAGE 39

26 next day, the cells were spun down and washed two times with 2xYT100 g/mL ampicillin (10mL per wash) and then resuspended in fresh media. The washed cells were used to inoculate 3 liters of fresh, autoclaved 2xYTg/mL ampicillin. Cells were grown with shaking (200rpm) at 37 C to A600 =0.5. The cells were then cooled to 2025 C in the cold room. The cells were then indu ced with a 0.4mM fina l concentration of isopropyl -thiogalactopyranoside (IPTG) and allo wed to express at room temperature (20-25 C) with shaking for an additional 8 hours. Cells were then harvested by centrifugation at 5000rpm for 30 minutes at 4 C using a JLA-200 rotor (Beckman). The cell pellets were drained thor oughly and were stored at -80 C until needed. The pellets can be stored at -80 C from at least 16 hours up to a w eek for maximum protein yield. Purification of Human Al kyladenine DNA Glycosylase 79 and Mutants The frozen pellets were weighed and re suspended on ice in cold buffer A (50mM sodium phosphate, pH 7.4, 1mM EDTA, 70 mM KCl, 10% glycerol, 0.5% 2mercaptoethanol, 1 g/mL pepstatin A, 1 g/mL leupeptin, 0.1 g/mL PMSF) Approximately 3mL of buffer per gram of pe llet was used. The resuspended pellets were then pooled into pre-chilled conical tubes and were lysed in a Aminco-SLM Instruments French press after loading into a 40K ce ll (30mL capacity) and pressing at medium (700psig) followed by maximum pressure (2550ps ig). The press was repeated again to ensure thorough lysing. The pressed cel ls were immediately placed on ice and centrifuged at 10, 000 rpm for 45 minutes in pre-chilled plastic tubes at 4 C using a JA20 rotor (Beckman). The supernatant, containi ng the soluble protein was then pooled into a pre-chilled conical tube a nd placed on ice. All further pu rification steps were carried out at 4 C and the protein fractions we re always kept on ice.

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27 The pooled supernatant was loaded on to a 200mL diethylaminoethyl (DEAE) cellulose (Sigma) anionexchange column us ing a peristaltic pump. The DEAE cellulose was batch equilibrated with several cha nges of 125mM sodium phosphate( pH 7.4) before packing until the material reached a pH of 7.48.0. It was then packed in a column and further equilibrated to pack and flow at a rate of 2mL/ min. using two column volumes of buffer A without the protease inhi bitors. After loading the supernatant on to the column, the column was washed with one column volume of buffer A with protease inhibitors. AAG which has a calculated pI of 9.1 will not bind to the DEAE and hence elutes in the wash. This step is useful in removing the va st amounts of DNA from the supernatant. The flow-through fractions were co llected in pre-chilled conical tubes placed on ice. They were then loaded at 2mL/ min onto a 5mL Hi-Trap sulfopropyl sepharose (Hi Trap S) cation exchange column (Amersham Pharmacia Biotech) using an Amersham Pharmacia Biotech FPLC system. The column was previously washed in 2 column volumes of 1M KCl to strip any protein stuck and then equilibrated in 5 column volumes of buffer A. AAG binds to the column under th ese conditions. An 80mL gradient from 70 to 500mM KCl in buffer A was then used to elute AAG from the Hi trap column. AAG usually eluted at approximat ely 250-300mM KCl. Fractions of 1.8mL were collected and stored on ice. The fractions eluting at 250300mM KCl were analyzed by SDS-PAGE for the presence of AAG and those frac tions that contain AAG were pooled and concentrated to 12mL in an Amicon U ltracentrifugation Stir red cell ( Model 8010) using a 5000D cut-off Polyethersulfone membrare, wetted in nanopure water. The concentrated fractions were then loaded on to a 125mL Sephacryl S200 HR gel filtration column, at 0.5mL/ min using an Amersham Pharmacia Biotech Gradifrac system.

PAGE 41

28 Previously, the column was isocratically de veloped at 1mL/ min with buffer B (25mM Tris, pH 7.5, 200mM KCl, 1mM EDTA, 1mM di thiothreitol and 5% v/v glycerol). Fractions were collected by washing the load ed fractions with buffer B. The fractions corresponding to the AAG peak were then analyzed by SDS-PAGE and the AAG containing fractions were pooled and concentrated as before to approximately 1.5 ml final volume. The purified, concentrated AAG wa s then dialysed against two changes of prechilled storage buffer( 50mM HEPES, pH 8.0, 100mM KCl, 1mM EDTA, 1mM dithiothreitol and 37.5% v/v glyc erol), in a 12, 500 cut-off dialysis membrane, for atleast 4 hours each. The final protein yield was determined by A280 measurements against a storage buffer blank using the published AAG extinction co-efficient of (27, 099 M-1cm-1). The dialyzed, pure AAG was dispen sed into prechilled eppendorf tubes as 810 L aliquots and stored at 80 C. The purity of the protein was assessed by staining a SDSPAGE gel with sypro-red dye. Typically, fresh aliquots were taken from the -80 C for an experiment. Any protein leftover from the used st ock was stored at -20 C and used within 3 days of storage. Serial dilutions of the stocks were done in cold AAG storage buffer prior to an experiment. Synthesis and Purification of Oligonucelotides Synthetic oligonucleotides were made on an ABI 392 DNA synthesizer using standard -cyanoethylphosphoramidite chemistry and reagents from Glen Research (Sterling, VA). The oligonucleotides were clea ved after synthesis from the solid support by treatment with fresh ammonium hydroxide an d were forced into collection vials with argon flush. The collected oligonucleotides cont ained base-protected products and other partial synthesis products. The protecting groups were removed by placing the vial into a

PAGE 42

29 55 C sand bath for 12-16 hours. The deprotecte d oligos were then concentrated to dryness in a Savant Hi-Speed centrifugal v acuum system. The dr ied oligonucleotides were resuspended in 50% glycerol for purification by denaturing PAGE. The samples were then resolved on a 12% polyacrylamide gel containing 8M urea, by loading around 4060 nM DNA per lane. Th e bands corresponding to the right sized product were visualized by UV shadowing and cut out from the gel. The DNA was eluted by shaking the gel slices in 8-15 mL of st erile NTE buffer ( 50mM Tris, pH 7.5, 50mM NaCl and 1mM EDTA), at room temperatur e. The buffer was changed every 8 hours three times and the eluted DNA was pooled. The pooled DNA was then passed through a 0.2m syringe filters (Acrodisc) to remove the gel remnants before dialyzing in a 3500 MW cut-off dialysis tubing (Spectropor) agai nst 4L of nanopure water, changing water every 8 hours, 3 times. The dialyzed DNA wa s concentrated to approximately 1.5mL final volume and then quantitated using A260 measurements and calculated extinction coefficients ( 94, 114 ). The oligonucleotides were then aliquoted and stored at -80 C. Radio-labeling of Substrates and Annealing to Complement DNA substrates strands cont aining the damaged nucleotide were 5end-labeled with [ ]32P-ATP using T4-kinase. A typical 50L reaction consisted of 2 M singlestranded 25mer containing the damaged base labeling buffer (50mM HEPES, pH 8.0, 100mM KCl, 6.4mM MgCl2), 30 Ci [ ]32P-ATP (Amersham) and 1 unit of T4 kinase (Invitrogen) at 37 C for at least one hour. The kinase was then inactivated by placing the tube at 95 C for 5 minutes. A 2-fold excess of the unlabeled complement was added to ensure complete annealing of all the labe led substrate. Annealing was performed by thoroughly heating the mixture to 85 C to remove all secondary structure and then slowly

PAGE 43

30 cooling to room temperature to aid the form ation of the most stable duplex. Typically 10% of the total DNA substrate was labeled when using a final DNA concentration of 2 M. When a final concentration of 200nM subs trate was used, with all other parameters remaining constant typically almost 100% of the substrate was labeled. For freshly labeled substrate, optimum times of e xposure for good signal ranged from 6-8 hours. Increasing times of exposure were used for older labels, ranging from 10-16 hours. Single Turnover Excision Ass ay for Glycosylase Activity To provide a measure of the chemistry of the enzyme, single turnover conditions in which a known amount of substrate DNA was inc ubated with a vast excess of AAG, were used. Single turnover conditions provide a meas ure of a step occurring after substrate binding and before product disso ciation, which in this case is presumed to be the chemistry step. Another reason for doing singl e turnover kinetics for assessing activity is that, in preliminary steadystate experi ments, it was observed that AAG could not recycle to excise all the subs trate present and hence seemed to be product inhibited in some way. Saturating conditions were used to assess glycosylase activity. Typically, either 50nM labeled duplex DNA (10% labe led) or 5nM labe led duplex DNA (100% labeled) were used as subs trate in the AAG assay buffer, consisting of 50mM HEPES pH 8.0, 100mM KCl, 0.5mM EDTA, 0.25mM DTT and 9.5 % v/v glycerol. At 37 C, the assay was started by adding AAG, typically anywhere from 4-to 20-fold excess AAG over substrate was used depending on the satu rating concentration for that particular substrate, which were determined empirically by titrations. At vari ous time points, from 30 seconds to 80 minutes, a 4 L aliquot of the reaction wa s withdrawn and quenched in 0.2M NaOH and chilled to stop the glycosylase activity. The rest of the assay relies on

PAGE 44

31 the fact that the abasic site after glycosyl ase action exists in equilibrium between the hemiacetal and the aldehyde forms of the de oxyribose. The aldehyde form is labile to base and heat and undergoes -elimination to yield a single stranded 5 labeled product upon denaturation, indicative of AAG mediated excision of the substrate base. After quenching and chilling, the t ubes were heated at 95 C for 10-15 minutes to ensure complete -elimination. The samples are then dilu ted with 2 volumes of loading buffer consisting of 95% formamide and 20 mM ED TA. Unreacted substrates were separated from cleaved products by PAGE on 12 % dena turing gels (8M urea, 1XTBE). The dried gel was exposed to a phosphor screen and visualized using a phosphorimager (Storm, Molecular Dynamics). Only the DNA with the 5-label is visible under these conditions, resolved between a faster migrating 13 mer product of AAG activity and a slower migrating, unexcised 25mer substrate. The ba nds were then analyzed using Image Quant software, which enables to quantify the -emissions. The number of counts is proportional to the amount of DNA in the band. The background radioactivity was controlled for by averaging the an alyzed bands with the counts in region of the gel which has no DNA. The product formed over time Pt was plotted over time using Kaleidagraph software. The plot was fit to a singlee xponential rise Equation3-1, using nonlinear regression. Pt = A0 (1e-kobst) (Eqn 3-1) Where A0 and kobs are the amplitude of product formed and observed rate of the exponential rise, respectively.

PAGE 45

32 Figure 3-2. A schematic of the excision assay and a sample gel for resolution of products from substrates. The glycosylase acti on, followed by base and heat exposure, causes complete -elimination to yield a 13-mer product, which is resolved on the gel from the 25-mer substrate. The 0 min time point is the no AAG control which also serves as the background c ontrol for spontaneous generation of basic sites during the assay. Multiple Turnover Assays for Glycosylase Activity Under multiple turnover conditions, concen trations of DNA (50nM) was in excess of AAG (1-20nM) were used. Other assay cond itions and procedures were the same as for the single turnover assays described a bove. To ensure total denaturation of the DNA after heating, twice the volume of Formamide/EDTA was used.. Electrophoretic Mobility Shift Assay (E MSA) for AAG binding activity Concentrations of labeled duplex DNA used to observe the binding properties of AAG were the same as used in the exci sion assays. Because, AAG was capable of 5 5 PAGE Analysis of Reaction Products 02482040 80 Time (min) 3 5 = 32P label 3 5 3 5 hAAG 0.2M NaOH 37 C 95 C

PAGE 46

33 excising the damaged base, a ca talytically inactive mutant of AAG, E125Q, in which the catalytic residue Glu-125 was mutated to a Gln was used. This mutant has identical binding properties to the w ild type AAG, but cannot excise the glycosidic bond. The EMSA consists of resolving bound versus fr ee species on a nondenaturing PAGE, to give a higher molecular weight, slower migr ating bound species and free, faster migrating species. Ideally, 50nM DNA was incubated on ice with 0 to 1600nM E125Q for 10 minutes and loaded on to a 6% non-denaturing PAGE, which was then run at 8V/cm for 180 minutes at 4 C, to prevent overheating of the fragile gel. When 5nM DNA was used, 0 to 160 nM E125Q was used. The dried ge ls were analyzed by phosphorimaging and ImageQuant software as in the excisi on assays to obtain free over bound DNA. DNA bound was plotted over concentration of E125Q and fit to a quadra tic equation (Equation 32) to get apparent binding constants (Kd) [EDtotal] = 2 40 0 2 0 0 0 0D E K D E K D Ed d (Eqn 3-2) Melting Temperature (Tm) Measurements For Duplex DNA Melting temperatures of duplex DNA substrat es were calculated using the Thermal program for DNA melting in the CARY-3 Bi o-UV-visible spectrophotometer (Varian Australia Pty Ltd., Australia). A temperature co ntroller (Peltier) was used to create a controlled rise in temperature. DNA substr ates containing the damaged base were annealed to equal concentrations of the co mplementary strand c ontaining T, F or C opposite the damaged base in 50mM HEPE S pH 8.0, 100mM KCl and 0.5mM EDTA, to a final duplex concentration of 4 M. Annealed DNA was diluted 6 times into masked cuvettes with caps to prevent evaporati on during melting. The dilution gave a final concentration of 0.67 M, which was determined to be the minimum concentration

PAGE 47

34 required to give an initial absorbance of 0.2, so as to be within the range of the BeerLambert law of absorbance. The temperature was increased from 35 C to 65 C at a rate of 0.5 C per minute. Tm values were calculated by taki ng the first derivative of the melting curve. Tm values correspond to the maximum value of the first derivative. Control experiments were done using te mperature increase rates of 0.25, 0.5 and 1 C per minute. Rates of 0.25 and 0.5 C per minute gave consistent results. Fluorescence Assay For Ethenoaden ine-AAG Binding and Excision A is intrinsically fluorescent( 94 ) and has an excitation max at 310nm with an emission max at 405nm (Figure 3-3). The fluorescent properties of A were used to monitor the binding and excision of AAG in real time. The fluorescence emission of A is considerably quenched when in double stranded DNA, but upon binding and excision of the glycosylic bond by AAG, fluorescence emi ssion is increased in intensity in a time based pattern indicative of AAG activity. Duplex DNA (100nM) containing A as the damaged base was incubated with saturating enzyme concentrations. For measur ing binding in the abse nce of excision, 4001600nM E125Q was used in standard AAG buff er, in which 0.5M HEPES was replaced by 0.5M Bicine(pH 8.0) as HEPES was found to be slightly fluorescent at the UV range. Replacing HEPES with Bicine did not affect AAG activity, as measured in 32P glycosylase assays. Binding re actions were carried at 25 C. To observe excision, AAG was used in the same concentrations as in the binding measurements and the reaction was carried out at 37 C. A fluorescence was monitored over ti me in a quartz cuvette. Data was collected using a Photon Technology Inc. QuantaMaster fluorimeter using a 75W xenonarc lamp. The band pass was set at 4nm with the excitation and emission

PAGE 48

35 monochromators set at 310nm and 410nm, respectively. Although the theoretical max for emission was found to be 405nm, reactions were monitored at 410nm to avoid the tryptophan fluorescence interf erence due to the protein at this wavelength. Each concentration titration was done as a se parate reaction. Buffer only and DNA only background signals were recorded for all spectra, before a dding the enzyme and starting the reaction. Figure 3-3. Fluorescent properties of 100nM A when in double stranded DNA and after excision by 400nM AAG at 37 C for 60 minutes. The blue and green spectra are excitation spectra, before and after excision by AAG, while the red and brown spectra are emission spectra, before and after excision b y AAG. The max for excitation and emission are given by the peaks ( see text ). Stopped-flow Fluorescence to Observe Flipping of A by AAG Since binding appeared to be too fast to measure by handmixing in the cuvette, stopped-flow measurements were done to obs erve binding and flippi ng in real time. DNA containing A, at a final concentration of 200nM, was added to AAG at a final

PAGE 49

36 concentration of 100, 200 or 400nM in standard AAG buffer at 20 C in a Biologic Stopped-flow fluorimeter. Excitation was set at 310nm and emission was measured in the 2-channel mode using 380nm cut on filters. Traces were taken for each reaction with 4 traces per channel, giving a total of 8 traces per mixing. Th e same number of traces was taken for the DNA only and AAG only cont rols for subtracting the backgrounds. Maximum number of traces was taken to increas e the signal to noise ratio as much as possible. These traces were then averaged to obtain the final signal, which was plotted against time to obtain a 5000millisecond time based binding curve. The data was not fitted, but the change in fluorescence intens ity was taken as a measure of binding and flipping of A.

PAGE 50

37 CHAPTER 4 EFFECTS OF HYDROGEN BONDING WI THIN A DAMAGED BASE PAIR ON THE ACTIVITY OF WILD-TYPE AND DNA -INTERCALATING MUTANTS OF HUMAN ALKYLADENINE DNA GLYCOSYLASE Structural studies of AAG ( 95, 96 ) and other DNA glycosylases have revealed that a nucleotide flipping mechanism is used fo r damaged base recognition and excision in which the damaged base is flipped out of the DNA helix and bound in an enzyme active site. In these nucleotide-flipped DNA glyc osylaseDNA complexes, an enzyme amino acid side chain is inserted into the base stack at the site vacated by the flipped base and may assist in nucleotide flipping by pushing th e damaged base from the helix. It is believed that DNA glycosylases actively flip damaged bases out of the helix rather than passively capturing bases that have transiently adopted extrahelical conformations. This active nucleotide flipping mechanism is supported by detailed kinetic studies of E. coli uracil DNA glycosylase which show a twostep binding mechanism where UDG initially binds DNA to form a non-flipped proteinDNA co mplex prior to flipping uracil from the helix ( 97 ). Many questions remain about how nucleotide flipping enables DNA glycosylases to discriminate between damaged and undama ged bases. For DNA glycosylases that have a narrow substrate specificity, a mechan ism in which a tight fit of the damaged base in the enzyme active site allows th e DNA glycosylase to discriminate between damaged and undamaged bases seems probable. For example, UDG excises only uracil from DNA and mutation of enzyme residues that form specific interactions with U alters the specificity of the enzyme so that it can excise C and T ( 96, 95 ). On the other hand,

PAGE 51

38 for DNA glycosylases that excise a structural ly diverse group of damaged bases such as AAG, a mechanism for damaged base recognitio n and excision that depends solely on specific interactions between enzyme binding pocket residues and a damaged base seems unlikely. Damaged bases excised, by AAG including 3-methyladenine, 1, N6ethenoadenine ( A), hypoxanthine (Hx), and 7-methyl guanine, have no obvious structural features in common that would allow the en zyme to distinguish between damaged and undamaged bases. In addition, the efficien cy of excision by AAG is dependent on the base pairing partner for some damaged bases ( 76 ) even though the enzyme makes no specific contacts with the base pairing partner in the crystal structures ( 77, 78 ). This base pair specificity of AAG further suggests th at substrate specificity is governed by a mechanism that involves more than the fit of the damaged base in the enzyme binding pocket. To further define the mechanisms of damaged base recognition and excision by AAG, the question of how nucleotide flipping contributes to the e fficiency of base excision by AAG was addressed using two gene ral approaches. First, site-directed mutations that were predicted to reduce the efficiency of nucleotide flipping were made to the DNA intercalating Tyr-162 residue of AAG. Second, hydrogen bonding interactions within the damaged base pair were removed by substitution of a nonhydrogen bonding partner, difluorotoluene (F), for thymine to increase the efficiency of nucleotide flipping by reducing the stability of the damage d base within the helix. Difluorotoluene is is oteric to thymine but lacks th e hydrogen bonding potential due to the substitution of the hydrogen bonding groups of thymine with elec tronegative fluorine (Figure 4-1). Kool and coworkers who desi gned F as a thymine analog found that it was

PAGE 52

39 a substrate of replicative polymerases and can be inserted into a gr owing strand just like thymine, making it a valuable tool for us e in replication and repair studies ( 105, 106 ). Since AAG substrates are damaged adenine bases encountered opposite T, F is a good reagent to use as a nonhydrogen bondi ng partner as it was a thymine analog. Experiments were designed to investigat e the effect of the Tyr-162 mutation on AAG activity and the effects of the DNA mutation in which T was replaced with F, on the activities of both wild type and mutant AAG. Figure4-1. Chemical structur es of hypoxanthine and 1, N6-ethenoadenine paired with thymine and difluorotoluene. F cannot form hydrogen bonds with Hx. Neither T nor F cannot hydrogen bond with A. DNA Substrates and Sequences The two AAG substrates used in these experiments were Hx and A. DNA duplexes were 25 nucleotides long, with the damaged bases in position 13, base paired with either T or F, with the rest of the sequence remaining perfectly complementary. The N N H O O C H 3 H N N H N N O H HxT N N H N N O F F C H3 H H HxF N N H N N N N N H O O C H 3 H AT H N F F C H 3 H N N N N AF

PAGE 53

40 DNA substrates were always duplexes, since AA G was found to be incapable of binding or excision on single stranded DNA. The s ubstrate DNAs were named based on the central damaged base and its base pairing partner, as Hx T, Hx F, A T or A F. The sequences of the DNA substrates are tabulated in Table 4-1. Table 4-1. Sequences of DNA substrates and positions of damaged base pairs Upper strand 5-GCG TCA AAA TGT NGG TAT TTC CAT G-3 (N= Hx or A) Lower strand 5-CAT GGA AAT ACC XAC ATT TTG ACG C-5 (X= T or F) Mutations to Tyr-162 and Projected Consequences To assess the contribution of the DNA-in tercalating Tyr-162 residue to the base excision activity of AAG, Tyr-162 was conve rted to Ser and Phe by site-directed mutagenesis to generate tw o mutant proteins, Y162S and Y162F, respectively. A catalytically inactive do uble mutant, Y162F/E125Q, was made for DNA binding experiments. Converting the Tyr-162 residue to Ser removes the aromatic ring generating a smaller amino acid side chai n that should not be able to penetrate the DNA helix as deeply when intercalated. Mutation of Ty r-162 to Phe removes the hydroxyl group but leaves the aromatic ring intact to intercal ate into the DNA base stack. These differences in insertion are illust rated in Figure 4-2 as cartoons in which Tyr-162 is replaced by Ser or Phe, as seen in the crystal structure. Figure 4-2 is not an actual st ructure but is meant to indicate possible differences in intercalation between Ty r-162 and the two amino-acids by simple replacement and not to indicate any other properties they may affect such as flipping. To see whether these in tercalating differences will be translated into flipping inefficiencies is the goal of this mutational analysis.

PAGE 54

41 Figure4-2. Projected differences in interc alation ability of Ser-162 and Phe-162 when compared to the wild type residue, Tyr-162, based on the crystal structure of AAG bound to A. Shown is a closeup view of the -hairpin (blue ribbons) and the intercalating residue (red spacefill) positioned in the helix (green ball and sticks) to fill the space vacated by the flippedout A (yellow ball and sticks) Ser and Phe may have different ab ilities to intercalate than Tyr (see text). Base Excision and DNA Binding Ac tivities of the Y162S Mutant Base excision activity for the Y162S mutant was measured in a chemical cleavage/gel assay for DNA substrates usi ng saturating AAG concentrations, in which excision of all the substrate is expected ba sed on previous kinetic studies. The strand containing the damage was end-labeled with 32P, prior to annealing to its complementary strand to create duplexes of otherwise identical sequences that contained HxT and AT base pairs. In 60minute assays using 1600 nM Y162S and 50 nM DNA substrate, no detectable base excision was observed for eith er DNA substrate. We estimate that the Y162S mutant is at least 1000fold less active than the w ild type AAG enzyme based on this result and using the conservative assu mption that 1 nM product (2% reaction) would have been detected if formed in these assays.

PAGE 55

42 The DNA binding activity of the Y162S muta nt was measured in electrophoretic mobility shift assays (EMSA) with the same damaged duplexes as used in excision assays, where the damage-containing DNA strand was 5-end-labeled with 32P. A damage-specific proteinDNA complex was not observed fo r the Y162S mutant with DNA substrates containing Hx or A opposite T (Figure 4-3). At high Y162S concentrations in the EMSA, a general sm earing of the DNA band was observed in a pattern similar to that for AAG with undama ged DNA (not shown). This smearing may represent weaker damage-independent DNA binding. Figure4-3. Electrophoretic mobility shift assays to measure the affinity of the Y162S mutant for DNA containing an AT or a HxT base pair. 50nM labeled, duplex DNA was incubated with 0800nM Y162S mutant. A band corresponding to a damage-specific prot einDNA complex is not observed for the Y162S mutant in assays with either an AT pair (left panel) or an HxT pair (right panel). Smearing of th e free DNA band is observed at 400 and 800 nM Y162S and may represent weak er damage-independent DNA binding. Free DNA [Enzyme]nM 800 800 0 0

PAGE 56

43 Activity of the Y162F Mutant Base Excision by the Y162F Mutant Single turnover kinetics of ex cision of Hx when paired with T was measured in a chemical cleavage/gel assay for both AAG and the Y162F mutant. Enzymes, at concentrations of 400, 800, and 1600 nM, in two separate experiments at each concentration, were incubated with 50 nM 32P-labeled 25-nt duplex DNA substrates at 37 C. Aliquots of each reaction mixture were withdrawn at several time points, quenched, and analyzed by PAGE to quantitate the concentra tion of products formed. For each enzyme, reaction time courses were essentially the same at all three concentrations demonstrating that single-tur nover conditions were met. Individual time courses were fit empirically to an expone ntial rise to calculate observed rates (kobs). Average values and standard deviations for kobs calculated from all si x experiments (two at each enzyme concentration) are shown in Table 4-2. Excision of Hx was 4-fold more rapid in assays with AAG than the Y162F mutant. The reaction course for 400nM enzyme is shown in Figure 4-4. Because AAG catalyzes excision of a st ructurally diverse group of damaged purine bases, the possibility that the Y162F mutation may ha ve differential effects on excision of different damaged bases was teste d. Kinetics of excision of the structurally dissimilar 1,N6-ethenoadenine placed opposite T were measured in single-turnover assays containing 400 and 800 nM enzyme. Reaction courses are shown in Figure 4-5. For each enzyme, observed rates were the same at both enzyme concentrations. The Y162F mutation had a smaller effect on the single turnover excision rate for A where AAG was 1.7-fold faster than the Y162F mutant (Table 4-2).

PAGE 57

44 To determine whether rates of excision of Hx would increase by making the base easier to displace from the helix, the T opposite Hx was replaced by difluorotoluene (F), which does not form hydrogen bonds with Hx (F igure 4-1). Single-tu rnover kinetics of excision of Hx opposite F was measured in th e chemical cleavage/gel assay with 50 nM DNA and 400, 800, and 1600 nM enzyme (Table 4-2). Excision rates were not dependent on enzyme concentration for either AAG or Y162F. Excision activities for both AAG and the Y162F mutant increased on th e HxF DNA substrate relative to the HxT duplex (data for 400 nM enzyme are s hown in Figure 4-4). The magnitude of the increase was greater for the Y162F mu tant (3.5-fold) than for AAG (2-fold). It is possible that the increased excision activity could be due to some effect of replacing T with F other than removing hydr ogen bonding interactions. To rule out this possibility, excision was also meas ured for DNA substrates containing AT and AF base pairs. A does not form Watson-Crick-type hydrogen bonding interactions with either T or F. Single-turnove r kinetics of excision of A opposite F were measured in chemical cleavage/gel assays using 50 nM DNA and 400 and 800 nM enzyme in separate experiments (Table 4-2). Th ere was not a significant e ffect on excision rates of A, as a 1.2-fold decrease in the excision rate for AAG and a 1.1-fold increase for the Y162F mutant were observed (Figure 4-5).

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45 Figure 4-4. Plots of time cour ses for Hx excision by wt AAG79 and Y162F Plots of the concentrations of abasic DNA produc t formed as a function of time in assays containing 400 nM enzyme and 50 nM DNA are shown. Because there is a relatively large difference in ra tes of Hx excision, time courses for excision of Hx paired with T (triangles) and F (squares) are plotted in separate graphs on different time scales for AAG (left) and theY162F mutant (right) Data plotted are average values from two independent experiments with standard deviations. Solid lines are the result of a single exponential fit to the data. 400nM AAG 400nM Y162F

PAGE 59

46 Figure 4-5.Plots of time courses for A excision by wt AAG 79 and Y162F The base pairing partner, T or F, did not affect A excision rates for either enzyme as demonstrated by overlapping time courses for excision of A by AAG on AT ( green squares ) and AF ( blue squares ) DNA and Y162F on AT ( red diamonds ) and AF ( orange diamonds ) DNA. Data plotted are average values from two independent experiment s with standard deviations. Solid lines are the result of a singl e exponential fit to the data. Results obtained from removing hydrogen bonding interactions to Hx indicated enhanced rates of excision of Hx by both the wt and the Y162F mutant. Normal purines are not usually excised by AAG. Experiments were done to see if any enhanced excision of normal purines opposite F was seen. In c ontrol experiments in which DNA substrates containing no damaged base, but ad enine or guanine opposite F ( A/G F), it was seen that removing hydrogen bonding interactions di d not make these normal DNA bases substrates for AAG. Similar AAG assays were performed to confirm this fact (Figure 46). Some groups have observed excision of normal bases by AAG at low levels, but we have not been able to demons trate this activity in the presence or the absence of hydrogen

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47 bonding interactions. The observa tions of these groups may be due to different exposure times of the gels or very long assay times. The nonexcision of normal purines opposite F goes on to further prove the fact that AAG uses flipping as an essential step in recognition but also coordina tes flipping with the active site parameters that decide what a substrate is. So though A or G could be made easier to f lip by putting opposite F, excision is not observed due to active site constraints. Table 4-2. Observed excision rates and re lative acitivitie s of AAG and Y162F mutant Enzyme Base pair kobs min-1a krel (AAG/Y162F)b Hx T 0.62 0.19 4.1 Hx F 1.3 0.1 2.5 A T 0.062 0.003 1.7 AAG A F 0.052 0.005 1.2 Hx T 0.15 0.03 Hx F 0.53 0.13 A T 0.037 0.001 Y162F A F 0.042 0.002 a,Values for kobs were calculated from single exponentia l fits to individual experiments. For Hx base pairs, two indepe ndent experiments were done at enzyme concentrations of 400, 800, and 1600 nM and average kobs values and standard de viations are reported for all six experiments. For A pairs, two independent expe riments were done at 400 and 800 nM enzyme and average kobs values and standard deviati ons are reported for the four experiments. b, Relative values, krel, were calculated from the ratio of the kobs for wt to Y162F for each base pair.

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48 Figure 4-6. Control glycosylase assay to show that base pa iring with F does not make A or G excisable by AAG. 100 minute time course gels are shown, with 50nM A F and G F DNA duplexes incubated with 800nM AAG at 37 C and quenched at the indicated time points. Th e C lane is the enzymeless control reaction. Only 25-mer substrates are seen, no 12-mer product, indicative of excision by AAG is seen on the gel. DNA Binding Ability of Y162F Mutant Mutation of the Tyr-162 residue to Phe re duces the DNA binding activity measured in electrophoretic mobility shift assays (EMSA). For and EMSA, 50 nM 32P-labeled duplex DNA substrates, identical to those used in excision assays, were incubated with increasing concentrations of enzyme (10 800 nM) prior to nondenaturing PAGE analysis. Since base excision would convert DNA substrates to products during the time course of EMSAs, catalytic ally inactive mutants (E125Q) of AAG and Y162F were used in these assays. The affinity of the Y162F/E125Q mutant for DNA containing a HxT pair is reduced relative to E125Q (Figure 47, upper panels). A concentration of 50 nM Y162F/E125Q is needed to form a similar fr action of enzymeDNA complex as seen with 50nM AF 50nM GF C 20 40 60 80 100 C 20 40 60 80 100Time(min)25 mer

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49 20 nM E125Q. At concentrations of 400 nM enzyme, about 70% of the DNA is bound by E125Q whereas about 25% is bound by Y162F based on the intensity of the bands. As reported previously ( 100 ), AAG binds to DNA containing an AT pair with greater affinity than a HxT pair (Figure 4-7 and Figur e 4-8, upper left panels). This is also true for the Y162F/E125Q mutant (Figure 4-7 a nd Figure 4-8, upper right panels). The Y162F/E125Q mutant binds DNA containing an AT pair more weakly than E125Q as it takes 20 nM Y162F/E125Q to form about th e same concentration of enzymeDNA complex as 10 nM E125Q. To determine what effect substitution of T with F would have on the DNA binding activity of AAG, assays were done fo r DNA substrates containing HxF and AF pairs. Binding assays contained 50 nM 32P-labeled duplex DNA and in creasing concentrations of AAG E125Q or Y162F/E125Q (10 800 nM). For both enzymes, binding was similar for DNA duplexes containing Hx T and HxF pairs (Figure 4-7, lower panels), and binding was slightly enhan ced on duplexes containing AF in comparison with AT (Figures 4-8, lower panels).

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50 Figure 4-7. Binding of wt AAG79 and the Y162F mutant to DNA containing HxT and HxF base pairs. Assays were done w ith duplexes containing either a HxT (upper panels) or HxF (lower panels) pa ir. Increasing con centrations (10 to 800 nM) of E125Q (left panels) and th e E125Q/Y162F mutant (right panels) were incubated with 50 nM DNA. Both enzymes were catalytically inactive mutants of 79AAG and the Y162F mutant, resp ectively. They were used so that binding efficiency ca n be observed in the absence of excision. Binding constants were not obtained due to the high degree of smearing which hampered exact quantitation.

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51 Figure 4-8. Binding of AAG and the Y 162F mutant to DNA containing AT and AF base pairs. EMSA assays were done as in Figure 4.6 with 25-nt duplexes containing either an AT (upper panels) or AF (lower panels) pair at position 13. Increasing concentratio ns (10 to 800 nM) of wt AAG (left panels) and the Y162F mutant (right panels) were incubated with 50 nM DNA. Catalytically inactive mutant s are used as in Figure 4.7. Implications of Flipping in th e Catalytic Efficiency of AAG The ability of DNA glycosylases to iden tify and excise damaged DNA bases is key to the overall success of base excision repa ir. Structural studie s of AAG reveal that

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52 flipping of the damaged nucleotide is the first step in substrate recognition, and this is facilitated by intercalating Tyr-162 into the space vacated by the flipped out nucleotide. In this study, the DNA-intercalating Ty r-162 residue of AAG was converted to serine (Y162S) and phenylalanin e (Y162F) by site-directed mutagenesis. A decrease in the base excision activities of both mutants was observed as expected if the Tyr-162 residue contributed to nucleo tide flipping by helping to pus h the damaged base from the helix. Base excision and damagespecific bi nding activities of th e Y162S mutant were reduced to undetectable levels for DNA substrates containing HxT and AT pairs, indicating that this mutant must be at least 1000-fold less active than AAG. The fact that DNA binding activity of the Y162S mutant wa s not detectable by EMSA suggests that the enzymeDNA complex seen for AAG is a nucleotide flipped complex. A similar mutation in UDG converting the DNA-intercalating Leu residue to Ala resulted in an 8 to 80-fold decrease in excision activity and mutation of Leu to Gly reduced UDGs excision activity by a factor of 100 600 ( 91, 92 ). The comparatively large effect of the Y162S mutation on AAGs activity may reflect a greater contribution of the DNA-intercalating residue to the activity of AAG than UDG. Mutation of Tyr-162 to Phe leaves the ar omatic ring to intercalate in DNA but removes the hydroxyl group from the aromatic ri ng. This mutation decreases the size of the DNA-intercalating residue much less than the Ser mutation but still affects the excision activity of the enzyme. Excision of Hx when paired with T by the Y162F mutant is 4 times slower than excision by the AAG and excision of A paired with T is 1.7 times slower. Interestingl y, the activity of the Y162F mutant is rescued on a DNA substrate where Hx is paired with F. The ex cision rate for the Y162F mutant increases to

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53 the rate measured for AAG excision of Hx pair ed with T. It is possible that making Hx easier to flip in the context of an HxF pair counterbalances a deficiency in the flipping ability of the Y162F mutant. An alternativ e explanation for the effect of the Y162F mutation on the excision activity of AAG is that the slightly smaller Phe residue is not able to push the displaced base as fa r into the enzyme binding pocket to align it properly for catalysis. If this were true then no difference in excision rates for Hx when paired with T and F would have been seen because the Phe mutant would have pushed Hx the same distance in both cases. The rationale for replacing T with F in Hx base pairs was that F is isosteric with T having the same overall shape but will not form hydrogen bonds with Hx. The expectation was that the lack of hydrogen bonding will increase the ease of flipping Hx by decreasing the stability of th e base pair. To rule out the possibility that F could have some other unanticipated effect on excision activit y, excision of A was measured when paired with T and F where neither pa ir forms hydrogen bonding interactions. Substitution of T with F had no signi ficant effect on excision rates of A for either AAG or the Y162F mutant whereas it increased the excision rate of Hx by a factor of 2 for AAG and about 3 to 4 for the Y162F mutant. T hus, the increase in Hx excision rates is likely to be due to changes in hydrogen bonding in teractions in the Hx pair. These results are consistent with a model where the ease of flipping a damaged base contributes to the base pair specificity of AAG. The kinetic mechanism for base excision by AAG is likely to contain a nucleotide flipping step in addition to the chemistry st ep where base excision occurs. Changing the ease of nucleotide flipping either by mutati ons to the enzyme or by changes to the

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54 stability of a damaged base within the he lix affects single turnover excision rates by altering the population of substrates stably flipped. The observation that substitution of T with F increases single tu rnover excision rates of Hx for both AAG and the Y162F mutant suggests that nucleotid e flipping is important for Hx excision. An altered flipping equilibrium for Hx excision would also expl ain the base pair specificity observed previously ( 75, 76 ). Two explanations are possibl e to explain why excision of A is not affected to a great degree by its base pairing partner. Either the nucleotide flipping step may not alter the flipping equilibrium as dras tically as for Hx, or the flipping equilibrium is not affected by the ba se pairing partner since A lacks hydrogen bonding interactions with its partner. A Two-step Selection Model for AAG Activity Based on these results th at were published ( 75 ) and previous work, we have developed a working model that explains the damaged base and base pair specificity of AAG. We propose that the specificity of base excision by AAG is governed by two important selection steps, nucleotide flipping and chemis try of bond cleavage which is affected by many factors such as proper fit of the base, alignment of the bond, suitability of the leaving group etc. in the enzyme active site. The enzyme may use the ease of flipping a damaged base as the initial crit erion for discriminating between damaged and undamaged bases and then use fit of the damaged base in the active site as a final check. The first nucleotide flipping selection step would be affected by changes in local DNA sequence or structure that affect the stability of a damaged base within the helix. Once a damaged base is flipped, it still must be a ligned properly in the act ive site for hydrolysis of the glycosidic bond to occur. This second criterion, proper fit in the active site, would explain why Hx but not G is excised fr om a wobble-type base pair with T ( 76 ). The 2-

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55 amino group may prevent G from fitting in the active site properly ( 78 ). The other factors in the active site may include suitability of the leaving group for chemistry, alignment of the glycosidic bond relative to the activated water and so on. An implication of this two step selection is that the overall efficiency of base excision repair may be a function of local DNA sequence and structure which affect the stability of damaged bases in the helix. A dependence on the efficiency of base excision on DNA sequence and structure could contribute to the formation of mutationa l hot spots and cold spots. Both AAG DNA-intercalating mutants and th e difluorotoluene base pair ing partner will be useful tools for testing this model further.

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56 CHAPTER 5 ACTIVITY OF HUMAN ALKYLADENINE DNA GLYCOSYLASE IS SENSITIVE TO THE LOCAL SEQUENCE CON TEXT OF THE DAMAGED BASE The contribution of nucleotide flipping to the excision efficiency of DNA glycosylases is not yet fully understood. Especially with re spect to a glycosylase like AAG, which has a diverse substrate range, the co ntribution of flipping could be critical to how the enzyme discriminates between substrat es and nonsubstrates and also how it can accommodate a diverse group of substrates. Th e contribution of flipping to substrate specificity could be unique for AAG since ma ny other glycosylases such as UDG act on a single substrate, in this instance, uracil on ly. But AAG acts on substr ates ranging from 3methyladenine and hypoxanthine (Hx) to 1, N6ethenoadenine ( A), which are not structurally related enough to enable the enzyme to use a common mechanism of recognition. Though the crystal structure of AAG does not indicate any specific contacts between the enzyme and the base pairing part ner to the flipped out damaged base, it has been shown by us that the excision efficiency of some damaged bases is dependent on the identity of the base pair and not the base alone ( 75, 76 ). The difference in excision can be directly related to the ease of flipping the damaged base in a given base pair because, removing hydrogen bonds which can make fli pping easier, also increased excision efficiency, in the case of Hx ( 75 ). This indicated that stability in DNA could affect repair of Hx by AAG. The minimum twostep m echanism for recognition and binding to the substrate discussed in the end of chapter 4 se rved as an additional guide to come up with novel methods to understand the contribution of flipping to Hx excision. The first step

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57 was to identify additional factors around Hx th at could contribute to its flippability by AAG. Since base stacking in DNA is a major stabilizing source, changes to the flanking neighbors to Hx were made, using either rela tively weak or strong base stacking partners. These may either further stabilize or destabili ze the Hx base pair in DNA and thus affect its flipping by AAG. To the base stacking ch anges were added additional modifications in the base pairing partner within th ese sequence contexts. This allowed for understanding more about how effects on ba se flipping would affect excision by AAG. DNA Substrates Indicating Base Stacking and Hydrogen Bonding Partners to Hx DNA substrates were designed to include base stacking changes around the central damaged base which was base 13 in the 25-me r long substrate. Hx was either flanked by T A base pairs (T Hx A) or G-C base pairs (G Hx C), the rationale being that, T-A and G-C nearest neighbors represent the weakest and strongest base st acking partners respectively ( 100 ). The G-C flanking base pairs may affect Hx accessi bility in DNA due to their intrinsically stronger base st acking. It has been shown prev iously that hydrogen bonding within the base pair affected excision of Hx by AAG presumably by increasing or decreasing the stability of Hx in DNA ( 75 ). So, Hx was base paired with T, F or C, within these sequence contexts. As shown in Figure 5-1, Hx has different hydrogen bonding interactions with these bases, forming a wobble base pair with T, no base pairs with F, and a Watson-Crick base pair with C. In contrast, A which is also a substrate for AAG, forms no hydrogen bonding interactions at all (Figure 5-1) and serves as a good control as seen with the AAG mutants (Chapter 4). Th e above sequences were incorporated into 25-nucleotides long substrates, with Hx at position 13. The rest of the DNA sequence was the same as the sequence shown in Table 4-1. Substrates used for experiments were 5-

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58 end labeled with 32P on the damaged strand and then annealed to the respective complement oligonucleotides. The DNA substrate nomenclatur e is based on the base st acking partners and the hydrogen bonding partners to Hx or A throughout this chapter. For example, a duplex substrate containing Hx with a 5T and a 3 A in which Hx is paired with T will be referred to as THxA T. Figure 5-1. Chemical structures of Hx and A base paired to thymin e, diflorotoluene and cytosine. Hx forms wobble base pairs with T, no base pairs with F and Watson-Crick base pairs with C. A forms no hydrogen bonds with any of the three base pairing partners. NNHOOCH3 H NNHNNOHHxT NNHNNOFFCH3HHHxF NNHNNNNNHOOCH3 HAT HN FFCH3HNNNNAF NNHNNNAC H-N NNHO H HxC NNHNNOH H-N NNHO H

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59 Base Stacking and Hydrogen Bonding Effects on Hx The flanking base pairs were designed to create strongest versus weakest base stacking interactions with Hx in DNA and he nce reflect on the ability of the enzyme to flip A or Hx. Base excision activity of AAG wa s measured using a chemical cleavage/ gel assay using labeled, double stranded DNA su bstrates with T-A or G-C base pairs flanking Hx, base paired to T. Increasing c oncentrations of enzyme (ranging from 20nM to 640nM) were incubated w ith 5nM substrate at 37 C. Aliquots were withdrawn at several time points from 0 to 80min., que nched, and analyzed by denaturing PAGE for product formation. For each enzyme concentrat ion, the reaction course was fit to an exponential rise to obtain observed rates ( kobs) which were used to compare excision efficiencies of Hx in the two sequence cont exts. Increasing concentrations of enzyme were used until no change in the progress of the time courses were observed, indicating that single turnover conditions were achieve d. For excision of HxT with G-C stacking partners, almost 4 times more enzyme was required to achie ve single turnover conditions compared to HxT with T-A stacking partners (Figure 5-2) and the observed rates at these saturating enzyme concentrations showed that excision of HxT was 1.6fold faster with T-A partners than with G-C ba se stacking partners. Rates ar e summarized in Table 5.1, at the end of the results section.

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60 Figure 5-2. Single turnover ex cision of Hx opposite T with T-A and G-C base stacking partners. With T-A base stacking partners ( left ), saturation was observed with 80 and 160nM AAG, whereas with GC base stacking partners ( right ), 640nM AAG was required to approach saturated excision of 5nM Hx T. Saturation required almost 4times more enzy me with the stronger base stacking partners ( See text ). Effects of Hydrogen Bonding Partners on Hx Excision in the St rong and Weak Base Stacking Context It is possible that making Hx less constrai ned in DNA may relieve the effects of the base stacking partners. This can be tested by replacing T with difl orotoluene (F), which does not form hydrogen bonds with Hx. In previous studies, removing hydrogen bonding to Hx was seen to improve its excision by AAG ( 75 ). In single turnover excision reactions, measured for 5nM HxF with both T-A and G-C base stac king partners with increasing concentrations of enzyme ( 20-160nM), it was seen that saturation was observed at relatively low enzyme concentr ations (Figure 5-3). At these saturating concentrations, when compared to the rates of excision of the same sequences with HxT, HxF excision was enhanced. The enhancement was 2-fold for HxF over HxT with T-A stacking partners but the enhancement was dramatic for Hx F over HxT with G-C

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61 stacking partners, making Hx F with G-C partners a be tter substrate than Hx F with T-A partners. The excision time course for Hx F with G-C partners was so fast that the early time points were not experimentally measurab le by hand mixing. It is presumed that the enhancement in excision rates is at least 10-fold over Hx T, as assumed from lower enzyme concentration excision assays. In cells during replicatio n, polymerases have a prope nsity to incorporate C, opposite Hx, with which it can form Wa tson-Crick hydrogen bonds, much like a G-C base pair ( 101-104 ). This preference makes Hx muta genic because with another round of replication, Hx C potentially becomes an AT GC transition. Given the observations with the effect of base pairing partners on Hx excision, it is importa nt to observe what effects base pairing with C will have on Hx excision, because even when WatsonCrick base paired with C, Hx is excisable by AAG. In previous experiments, it was shown that Hx excision was slower opposite C than opposite T ( 76 ). To determine how base stacking partners can affect Hx excision opposite C, single turnover excisi on reactions were done with 5nM HxC with T-A and G-C base st acking partners using 160 and 320nM AAG. Under these conditions, kinetics of excision of Hx was very slow (Figure 5-4). Notably, when base paired to C, Hx excision was una ffected by the base st acking partners, unlike when opposite T or F. Excision of HxC in both sequence contexts essentially proceeded at the same rate (Table 5-1).

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62 Figure 5-3. Single turnover ex cision of Hx with T-A and G-C base stacking partners opposite nonhydrogen bonding base pair ing partner, F. Excision of Hx F was much faster than opposite T. In creasing AAG concentrations of 20nM ( blue squares ), 40nM ( yellow triangles ), 80nM ( green squares ) and 160nM ( pink circles ) were used. With TA base stacking partners, 5nM Hx F was excised to saturation by only 40nM AAG ( top, yellow triangles ). But with 5nm Hx F with G-C base stacking partne rs, excision was fastest, with saturation achieved by only 20nm AAG ( bottom, blue squares ). A shorter time scale has been shown in these gra phs when compared to Figure 5.2, to emphasize the faster excision achieved for these substrates.

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63 Figure 5-4. Single turnover ex cision of Hx with T-A and G-C base stacking partners opposite Watson-Crick hydrogen bonding partner, C Excision of 5nM Hx C with T-A base stacking partners did not saturate with 160nM ( blue squares ) or 320nM ( red triangles ) AAG. The same trend was seen with G-C stacking partners with 160nM ( green squares ) and 320nM ( pink circles ) AAG. Time courses for Hx C excision in both sequence contexts closely mirror each other, indicating that excision of Hx was not affected by base stacking partners when Watson-Crick base paired with C. Effects of Base Stacking Partners on Binding to Hx Substrates by AAG It is possible that G-C base stacking partners made fli pping Hx less favorable than T-A base stacking partners, by increasing it s stability in DNA. The higher molecular weight band observed in our EMSA is indi cative of a flipped ba seenzyme complex, which would give a lower intensity shifted ba nd with G-C stacking partners than with TA stacking partners. Lesser intensity shif ted bands were seen with the AAG mutants incapable or less capable of flipping the damaged base ( 75 ). To test this conclusion, 5nM 32Plabeled substrates, identical to the ones used for excision assays, were incubated with

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64 0-80nM enzyme and an EMSA was performe d to separate free DNA from the higher molecular weight enzymeDNA complex, indica tive of specific bindi ng. A catalytically inactive mutant of AAG, E125Q was used in binding assays, to overcome the problem of loss of substrate due to excision by AAG during the time course of the EMSA. The binding affinity of the enzyme for HxT with T-A base stacking partners was higher than with G-C base stacking partners. With TA base stacking part ners, at 80nM E125Q, almost all the substrate around 90%, was bound by the enzyme (Figure 5-5A, upper panel ) whereas, with G-C base stacking partners binding was less efficient and even at 80nM enzyme, only about 50% of th e substrate was bound (Figure 5-5A, lower panel ). This is also consistent with the observation that more enzyme was required to saturate GHxCT in excision assays. Effects of Hydrogen Bonding Partners on Binding to Hx in the Strong and Weak Base Stacking Context The same enhancement shown with excisi on of HxF was also seen in improved binding affinity of E125Q to both substrates in assays performed w ith 5nM substrate and 0-80nM E125Q. The enzyme-DNA complex band appeared at lower E125Q concentrations than when opposite T with T-A stacking partners (Figure 5-5B, upper panel ). With G-C base stacking partners, al most all the substrate around 90% was bound by 40nM enzyme with no free substrate det ectable with 80nM enzyme (Figure 5-5B, lower panel ). This is much improved binding when compared to HxT in the same sequence context, in which only 50% of the substrate was bound by 40nM enzyme (Figure 5-5B, upper panel The improved binding affinity was more pronounced for the G-C substrates than for the T-A substrates, as was observed with excision efficiency.

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65 Binding affinity of the enzyme for HxC mirrored the excision efficiency. Duplex substrate (5nM) was incubated with 0-320nM E125Q and then resolved by EMSA. But binding affinity was very weak fo r both DNA substrates (Figure 5-5C, upper and lower panels ). A specific enzyme-DNA complex band was hardly visible even with 320nM E125Q. Figure 5-5. Electrophoretic mob ility shift assays to m easure binding of AAG to DNA containing Hx in diffe rent sequence contexts Binding of E125Q to 5nM duplex DNA was observed as the appearan ce of the higher molecular weight band in EMSAs. Binding to Hx opposite when flanked by T-A stacking partners ( upper panel ) and G-C stacking partners ( lower panel ), with T ( A ), F ( B ) or C ( C ) base pairing partners are shown. ( See text )

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66 Base Stacking and Hydrogen Bonding Effects on A Effects of Base Stacking and Hydrogen Bonding Partners on A Excision by AAG In previous experiments it was seen that substitution of F for T opposite A did not affect A excision rates. We proposed that this was due to A not making hydrogen bonding interactions with either T or F. Gi ven the large magnitude of enhancement in excision seen for HxF with G-C base stack ing partners, it woul d be interesting to determine whether base stacking part ners may alter the effect of F on A excision. Single turnover excision of AT and AF, both with G-C base stacking partners were measured for 5nM labeled substrate and 20nM -320nM AAG. There were few differences in the time courses, and rates of excision of A was largely unaffected by substitution of F for T (Figure 5-6). The rate enhancement was 1.2-fold of AF over AT, which is similar to the rate enhancement observed for AF over AT when flanked by T-G base pairs ( 75 ). Excision of A was much slower than excisi on of Hx, irrespective of the sequence context, highlighting the fact that AAG had a diverse substrate range which may differ intrinsically in their interaction with the enzyme. A excision opposite C was also similar to excision when opposite T and F with G-C stacking partners (Figure 5-7). In all three sequences A excision kinetics followed the same time course and showed similar rates of excision (Table 5-1). Striki ngly, excision of Hx opposite C with both T-A and G-C stacking partners resemb led the kinetics of excision of A in any sequence context.

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67 Figure 5-6. Single turn over excision of A with G-C stacking partners. 5nM A T (left) and A F (right) were used in excision assays with 20 to 160nM AAG to acheve single turnover conditions. These sequences were used as controls because A excision is not affected by base pairing partners. The dramatic effects seen for Hx excision with G-C stacking partners was not observed for A excision, the time courses for both A T and A F substrates when with G-C stacking partners were very similiar. Figure 5-7. Single turn over excision of A opposite C with G-C base stacking partners. 5nM A C was excised by 80 and 160nM AAG. The time courses and rates were similar to those observed for A T and A F.

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68 Effects of Base Stacking and Hydroge n Bonding Partners on Binding to A Substrates by AAG No effect was seen on binding affinities to AT and AF flanked by G-C base pairs, in EMSAs (Figure 5-8A and B), requi ring only 10-20nM enzyme to bind all of the substrate in both cases. Binding to AC DNA was as efficient as binding to the other two A substrates (Figure 5-8C). Figure 5-8. Binding of E125Q to A substrates with G-C stacking partner s. Binding of 080nM E125Q to 5nM A T ( A ), A F ( B ) and A C ( C ) was observed in EMSAs. Binding affinity to all three A substrates was essentially the same, with almost all DNA bound by 10nM E125Q. Melting Temperatures of Hx and A Substrates The effects of base stacking and hydroge n bonding partners on the excision of Hx could be predominantly due to altered stab ility of Hx in the DNA in these sequence contexts, which in turn would reflect on how efficiently AAG can bind and flip Hx. These effects were seen in the amount of enzyme required to br ing about saturated excision and the binding to DNA in different sequence contexts. To determine if base Enzyme-DNA complex [Enzyme]nM Free DNA

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69 stacking and hydrogen bonding partners bring about altered stability in DNA, melting temperatures were measured by monitoring th e change in UV absorbance at 260nm over a controlled temperature ramp of 0.5 C per minute. A melting curve was obtained whose first derivative was taken to gi ve a peak corresponding to the Tm. Mean Tm with standard deviation from two independent experiments for each substrate are given in Table 5-1. As a general trend, it was seen that the Tm for substrates with Hx and A opposite C were the highest when compared to the same bases oppos ite T or F, within the same flanking base pairs. For Hx with G-C base stacking partners, the Tm was highest when base paired with C, intermediate when base paired with T and lowest when base paired with F. Generally, all three melting temperatures were higher than the corresponding ones for Hx with T-A base stacking partners. So it can be conclude d that, the strong base stacking partners, may affect the stability of Hx considerably and hence the added effects of hydrogen bonding or removal of hydrogen bonding are more eviden t in this sequence context. This also agrees well with the excision rates of Hx in these sequence contexts. When placed between T-A base stacking neighbors, differences in Tm are not as distinct as for G-C neighbors. But the differences in Hx excision rates were also mode st in this sequence context when T was replaced by F. A did not show any differe nces in excision when T was replaced by F, and Tm for the A substrates are not very vari ed. It must also be noted here that A excision is much slower than Hx ex cision and may be mechanistically very different from Hx excision. He nce it is hard to correlate A excision with the observed Tm and compare them with the other Tm for Hx substrates. It was also seen that for Hx with T-A base stacking partners and the A substrates, the melting temperature was slightly higher when T was replaced by F. This may be because; F is a good stacker in DNA ( 105,

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70 106 ). This stacking property of F may not a ffect the flipping eff ects due to lack of hydrogen bonds, but may just affect the Tm and hence the observation that for F in these two sequence contexts, reduced Tm may not necessarily imp licate increased excision rates. Table 5.1. Melting temper atures of Hx and A substrates and corresponding single turnover excision rates Substrate Base pairing partner Tm ( C) kobs (min-1)* T 45.5 0.0 1.5 0.3 F 46.5 1.0 2.6 0.4 THxA C 48.3 1.3 0.07 0.01 T 51.5 0.5 0.95 0.07 F 47.8 0.3 nd GHxC C 53 0.0 0.06 0.01 T 47.0 0.5 0.04 0.00 F 48.5 0.0 0.07 0.00 G AC C 49.3 0.3 0.075 0.007 kobs values are based on mean single tur nover excision rates for two independent experiments at saturation, w ith standard deviations. kobs values for GHxC opposite F were experi mentally too fast to measure by hand. ( See text ). Sequence Context Effects and Im plications for AAG Activity The active flipping mechanism of DNA glyc osylases is very important for the overall efficiency of the base excision repair pathway. In the case of AAG, though flipping is facilitated by the intercalation of Tyr-162 into the helix, how the damaged base and its interactions in DNA can contribute to recogniti on, makes an in teresting line of thought. Though the crystal structures of AAG, bound to a pyrrolidine abasic site analog ( 77 ) and 1, N6ethenoadenine ( 78 ) show no specific contacts between the enzyme

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71 with the base opposite the damaged base, excisi on efficiency of certain damaged bases is more an identity of the base pair than the base itself ( 76 ). It has been shown that AAG substrates vary in their sensitivity towards the base pairing partne r and mutations to Tyr162, which may decrease flipping efficiency ( 75 ). Only for Hx, removing hydrogen bonding could rescue the Y162F mutation, to th e level of activity of the wild type enzyme. No rescue occurred for A. This is a reminder of th e diverse substrate range of AAG and that different steps in recognition and excision contri bute to different levels for the substrates. The thymine analog, difluor otoluene, indicated that increasing the flexibility of Hx in DNA will also increase its fli ppability by AAG. Added to the effect of the base pairing partner, flippa bility can potentially be affected by other changes around the damaged base such as base stac king partners. Base stacking partners may increase or decrease the st ability of base in DNA. In this chapter, the 5 and 3 base stacking partners to Hx were chosen as possible candidates to change and thereby affect flippability of Hx by AAG. Either T-A base pair s or G-C base pairs were chosen as the base stack ing partners to represent re latively weak and strong base stacking neighbors to Hx. A working model fo r the activity of AA G has already been discussed based on the results from Chapter 4, in which the enzyme can use a twostep process to specifically identify and excise its substrates efficiently. In this model, the enzyme uses the flipping step as an initial test for destabilized bases in DNA, which could indicate possible substrates. After the base is flipped, AAG uses the factors in the active site like proper fit in the active site, al ignment of the glycosylic bond for cleavage and suitability of the leaving group that lead to chemistry to finally identify the flipped base as a substrate and excise it from DNA. A ccording to this model, flipping can be an

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72 important determinant of the enzymes efficien cy. Factors that affect the flippability of a base may in turn contribute to excision efficiency too. It required about 4 times more enzyme to saturate the rate of excision of HxT with G-C than T-A stacking partners and excisi on was also 1.6-fold slower than for TA neighbors. This shows that the strong base stacking contributed by the G-C base pairs probably increased the stability of HxT and made it much more difficult to be flipped by the enzyme, as can also be seen in the reduced binding affinity. If this were the case, then removing the constraint of hydrogen bonding must be able to dest abilize Hx more and hence make it more easily flipped. This is exactly what happened when T was replaced by F, to which Hx cannot hydrogen bond. Both binding affinity and excision efficiency increase for HxF, in both sequences. Most striking was the enhancement of excision rates seen with G-C stacking partners, which was a 9-fold enhancement, compared to a 2fold enhancement with T-A stacking partners The stronger base st acking partners, G-C base pairs, seem to have exaggerated th e effect of F on Hx excision by AAG, possibly owing to the fact that the e ffect of destabilization of Hx F on flipping was more pronounced when in a stronger base stacki ng sequence. This could mean that the interactions between AAG and the DNA during the formation of a stableflipped Hx complex were stabilized by the strong base stacking partners while at the same time, F destabilized Hx to enable eas ier flipping by AAG. The weaker base stacking sequence, TA partners, may not have the same effect on the enzymes interaction with DNA, and hence the base pairing partne r effects on flipping were mode st. The assumption that DNA stability is affected by the base stacking pa rtners is backed by the melting temperatures measured for the various substrates.

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73 In contrast, A excision was not greatly altere d when placed between G-C base pairs. The rate of A excision and the binding of AAG to A substrates when placed between G-C stacking partners mirrored the rates and binding properties of A placed between T-G partners ( 75 ). It has been shown before that A excision was not affected by the base pairing partner. Binding was unaltered too. Since A lacks the ability to hydrogen bond with any base, the base pairing pa rtners did not add to its destabilization in any way. It is also possible that its intrinsic instability in DNA makes A adopt an extra helical conformation much more easily than Hx; hence making it more accessible to AAG. For Hx the wobble base pair with T adds to its stability and impedes flipping, which can be overcome by other changes that decrease stability. This was seen when Hx is placed between T-A base pairs and when placed opposite a non-hydrogen bonding partner. Strikingly though, this neighboring base pair sensi tivity was lost when Hx was Watson-Crick base paired with C. In th e cell, Hx is formed opposite T due to deamination of A but; replicative polyme rases have a high propensity to place a C opposite Hx, which leads to transition mutations. The fact that, when opposite C, Hx repair is impaired to the same degree irresp ective of the base stack ing partners indicates an important biological function. Hx repa ir opposite C would be mutagenic and a Watson-Crick base pair may overcome the base stacking partner effect to prevent repair. So stability of Hx in DNA may be an importa nt factor in deciding its fate, a wobble base pair may make it more discernible by AAG while a WatsonCrick base pair may make it less discernible, possibly by shifting the flipping equilibrium. This difference in stability was partly reflected in the melting temperatures for the various substrates, especially for

PAGE 87

74 the Hx base pairs between GC base pairs, with HxC substrates having the highest Tms and HxF substrates having the lowest Tms, with HxT being in between. Only a modest difference in Tms was noticed for the Hx base pairs with T-A stacking partners, adding weight to the argument that base stacking partners can affect the stability of the Hx base pair in the substrates. An important biological outco me of this scenario, in which local sequence context is seen to affect Hx removal by AAG, is its significance in understa nding the presence of mutational hot-spots and cold -spots in the genome. The effect of local sequence context in the stability of Hx in DNA can play a major role in both enhancing and reducing its repair. This in turn may contri bute to the complex interplay between factors dedicated to protecting the cell from mutations, like repair mechanisms and the limitations facing them.

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75 CHAPTER 6 ACTIVITY AND STABILITY OF AAG DURING ASSAYS The kinetics of AAG revealed a puzzli ng dilemma. The enzyme was not able to catalyze multiple rounds of excision. Under multiple turnover conditions, AAG performed anywhere from one to three turnov ers and then seemed to stop catalyzing base excision. Under multiple turnover conditions, th ere is excess substrate over enzyme and ideally, an enzyme goes on catalyzing until all the substrate is depleted. But under these conditions, factors such as di ssociation from product and rea ssociation with substrate will affect the catalysis by the enzyme. So single turnover conditions in which product dissociation will not contribute to the observed rate of catalysis were used in all experiments to study the properties and mech anism of action of AAG. Under single turnover conditions, there is a vast exce ss of enzyme over substrate and ideally, saturation of substrate is observed. The observe d excision rates are a measure of the rate of excision when substrate is saturated with enzyme and reflects the rate of some step after binding the substrate and prior to product dissoci ation. Although si ngle turnover excision kinetics offers a good measure of AAGs activity, the fact that the enzyme cannot catalyze several rounds of excision pr ompted several questions relating to its activity and stability. Is the enzyme losing activ ity during the experiment? Is this property also a function of the substrate and its se quence context? Are r eaction conditions not ideal for AAG? These were important questions to address in order to design experiments in the future and make the most of the knowledge gained from previous experiments. In addition, evidence was mounting about similar activities observed for other glycosylases

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76 like human thymine DNA glycosylase ( 107 ) and the human MutY homolog ( 108 ). These questions, when answered may reveal some missing links between glycosylase activity and probably, the BER pathway and its components. So, a systematic experimental design to address these questions was performe d. In these experiments, enzyme death, the turnover issue as an effect of sequences and other conditions th at could improve AAG activity were considered as possible candidates to test. Loss of AAG Activity Can Cont ribute to Reduced Catalysis AAG loses activity when stored be low a final concentration of 40 M for periods longer than 6-8 months at -80 C. The enzyme also lost activity, both binding and excision, when stored for more than 10 days at -20 C. This indicated that, either the enzyme had to be stored above a critical conc entration to preserve activity or the enzyme slowly loses activity over time, whatever conc entration it may be. This loss of activity might be more significant at the higher temperature in which excision assays were performed. To determine if this is true, two simple enzyme death assays were performed. In both assays, 5nM THxA T was used as the substrate with 40nM AAG. This was the substrate of choice because Hx was found to be efficiently excised by AAG but excision was not so fast that hand mixing experiments could not be used (Chapter 5). For single turnover excision assa ys, a 4X concentration of the enzyme was needed for the assay stored in ice and diluted directly into the assay mix to start the reaction. In the first enzyme death assay, 160nM AAG was pre-in cubated for times ranging from 0 to 120 minutes at 37 C. Excision assays were performed to determine the loss of activity during the pre-incubation. Time points were take n as for a normal excision assay and the quenched samples were analyzed by PAGE and the product formed plotted over time

PAGE 90

77 (Figure 6-1). The products formed and the ra te of product formation was compared for various periods of pre-incubation at 37 C (Table 6-1). It was dete rmined that the loss of activity was not drastic. When compared to th e No pre-incubation control, there was a slight drop in the amount of product forme d, progressively, with increasing times of incubation, from 4.8nM to 4.4nM. However the ra tes of excision did drop with increasing pre-incubation times, to almost two times lower than the control rate (Table 6-1). Even after 2 hours at 37 C, the loss of activity was much less than the activity of AAG on the slow substrates, for example, Hx C for which, 160320nM AAG was required to observe comparable activity. Figure 6-1. AAG death assay under singl e turnover conditions. 5nM THxA T was excised by 40nM AAG diluted from a 160nm AAG stock which was incubated at 37 C for the indicated times (0 to 240 minutes). Reactions were started by diluting the stock 4-times in to the tube. A serious loss of AAG activity was not seen even after 240 minutes at 37 C.

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78 Table 6.1. Comparison of pre-incubation of 160nM AAG at 37 C with product formed and rates of product formation with 5nM THxA T Pre-incubation at 37 C (min) Product formed (nM) kobs (min-1) 0 4.8 0.4 30 4.5 0.3 60 4.5 0.3 120 4.4 0.25 240 4.4 0.2 As there was not a significant loss of enzyme activity upon incubation of the 4X stock at 37 C, another approach was chosen, in which AAG was pre-incubated at 37 C with the assay buffer for the same time periods used before, but at a final assay concentration of 40nM instead of 160nM. Th e excision assay was started by adding 5nM substrate to the pre-incubated AAG. This will also mimic the condition of the usual assay carried out and may tell us more about wh ether AAG was losing activity during the assay itself. So, AAG was pre-incubated at 37 C for times ranging fro m 0-240 minutes and then the reactions were started by adding DNA to each mix, to give a final enzyme concentration of 40nM and substrate con centration of 5nM. The experiment was performed as before and product formed (Fi gure 6-2) with the si ngle turnover excision rates (Table 6-2) was obtained. In this case, the observed loss of activity was considerable. Both the amount of product formed as well as the rate of product formation was reduced when compared to the No pr e-incubation control. The progressive reduction was more pronounced for 40nM AAG than for 160nM AAG. In 60 minutes at 37 C, both the amount of product and the rate were only one-third of the No preincubation control. After 240 minutes of pr e-incubation, there was no detectable product

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79 formed. This means that the enzyme was totally inactive after this extended period at 37 C. However a significant drop occurred with every pre-incubation time at 37 C beginning at 30 minutes (Table 6-2), suggesting that AAG c ould be progressively losing activity during the time course of our single turnover assays. This may either be due to enzyme instability or the absence of an additiona l factor that could stab ilize it. This factor may range from the right pH or i on to another specific protein. These death assays were an important i ndication of the limitations faced in our experiments, the enzyme itself. The additional concentration-dependent loss of activity highlighted how enzyme death may be more evident when less AAG was used. Consequently, there could be a critical concentration below which enzyme death could be rapid. In the traditional multiple turnover assa y with an excess of substrate over enzyme, this phenomenon may be contributing significantl y to the lack of turnovers. According to Selwyn (115) the test for enzyme inactivation duri ng the course of an assay comes from the super-imposability of the progress curves at any concentration of the enzyme. When the enzyme loses activity over the progress of the assay, the enzyme itself will follow a firstorder time dependence varying with c oncentration. Hence the product formed over time may vary with different enzyme c oncentrations, giving nonsuper-imposable progress curves. This property due to enzyme inactivation is called the Selwyns test. The behavior of AAG at the two different co ncentrations used, along with the progress curves obtained during single tur nover titrations (Chapter 5) indicate similarities with the behavior under Selwyn test. This similarity may indicate possible loss of AAG activity during the assay and needs to be further inve stigated for designing st rategies to explain the inactivity and to overcome the effects of inactivity.

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80 Figure 6-2. AAG death assay unde r single turnover conditions Assays were done with 5nM THxA T and 40nMAAG which were preincubated at 37 C for the indicated times (0 to 240 minutes). Reac tions were started by adding substrate to preincubated AAG. A considerable lo ss of AAG activity was seen starting at 30 minutes to no detectab le activity after 240 minutes.

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81 Table 6.2. Comparison of pre-incubation of 40nM AAG at 37 C with the product formed and rate of product formation with 5nM THxA T Pre-incubation at 37 C (min) Product formed (nM) kobs (min-1) 0 4.1 0.3 30 3.8 0.2 60 3.5 0.1 120 2.0 0.1 240 Not detectable Not detectable Multiple Turnover of Hx Is Dependent on the Base Pairing Partner Based on the previous results which reve aled base pairing and base stacking sensitivities in AAG mediated excision of Hx, it was clear th at Hx excision depends more on local sequence context than A. Under multiple turnover conditions, using a THxG substrate with T being the base pairing pa rtner to Hx, approxima tely two or three turnovers were always observed. This mean t that, AAG was able to catalyze more than one enzyme equivalent, but is limited by othe r factors and then is not able to catalyze excision of all the substrate. Removing hydr ogen bonding to Hx was seen to improve its excision, especially with strong base stacking partners. A multiple turnover excision assay, in which 50nM Hx with GC stacking partners was used, opposite either T or F with 5nM AAG was performed. When opposite F, almost 45nM substrate was excised completing about 9 turnovers for AAG. On th e other hand, the same substrate opposite T showed just 3nM substrate excised, meaning that AAG was unable to complete more than half a turnover (Figure 6-3). This was even less than the usual numb er of two or three turnovers seen for Hx T substrates with TG base stack ing partners. This inhibition of turnovers for Hx with GC stacking partners opposite T was observed again when 20nM AAG was used with 50nM substrate, while the same substrate opposite F was always

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82 fully excised. In multiple turnover experiments done with the previous THxG substrate, which should be intermediate between TA and GC stacking sequences, F as the base pairing partner stimulated complete excision of Hx. Whereas T as the base pairing partner gave about two or thr ee turnovers (Figure 6.4). Figure 6-3. Multiple turnover of Hx T and Hx F with G-C stacking partners. 50nM substrate and either 5 or 20nM AAG were used in these assays. Hx F was turned over completely by both concentr ations of enzyme, meaning that AAG was able to do multiple turnovers of Hx when not hydrogen bonded. On the other hand, excision of Hx T showed only half a turnover. This restriction on AAG turnovers seemed to be sensitiv e to the hydrogen bonding partner.

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83 Figure 6-4. Multiple turnover of Hx T and Hx F with TG stacking partners. 50nM substrate and 10nM AAG were used. In the presence of stacking partners, intermediate between TA or GC base stacking partners, Hx T excision showed around two or three tu rnovers with AAG, whereas, Hx F was excised to completion by AAG. There seemed to be a clear connection betw een the stability of Hx in DNA and the ability of AAG to turnover Hx. This could m ean that the extremely fast excision of Hx F in any sequence context is over shadowing th e combined effects of loss of AAG activity and any other modes of inhibi tion possible in a multiple turnover reaction. A multiple turnover reaction may reflect produc t interactions with the enzyme that are not a factor in a single turnover reactio n. So, the differences in turnover seen for Hx depending on the sequence context may be an exaggeration of the enzyme being slow due to the strong base stacking and hydrogen bonding sequen ce context and at the same time losing activity rapidly. It must also be recalled that in the st ronger base stacking sequence, binding is much less efficient. The sequence context of the substrate DNA may play an

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84 important role in taking into account produc t inhibition issues, which may make much less enzyme available to bind the substrat e in addition to reduced binding affinity. Multiple and Single Turnover of A Present Different Pictures Sequence context had little effect on the excision of A. Whatever may be the base stacking or base pa iring partners to A, under single turnover conditions, product formed and the rate of product formation were vi rtually unchanged. This same behavior was observed under multiple turnover conditions t oo. Whatever the sequence context, exactly one equivalent of the amount of AAG used was excised when A was the substrate. Whether it was A opposite T or F, only one turnover was seen using G AC DNA. 50nM substrate and 5 or 10nM AAG were used in multiple turnover reactions as described above. It was surprising that compared to th e progress of singleturnover reactions, the burst of product to reach one turnover was much faster under multiple turnover conditions. Therefore, a titration of AAG to span multiple turnover and single turnover conditions was done with 5nM A T with GC stacking partners, the same substrate used before. It was seen that the lower the enzy me concentration, the faster the burst of product, though under multiple turnover condi tions, only one turnover was seen (Figure 6-5). As the AAG concentration reached singl e turnover levels, above 5nM, the product formation curve resembled previous single tu rnover progresses and the rates of single turnover were much lower than multiple tu rnover. It was also observed that, as AAG concentration increased in the multiple tu rnover part of the titration, rates dropped whereas, in the single turnover part of the tit ration, rates remained constant throughout the concentration range (Table 6-3).

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85 The above observations appeared to conf lict with those made previously by Dr. Clint Abner, who conducted a similar titration with 50nM A DNA and 10800nM AAG, final concentrations. He observed a concentration depende nt rise in product formed consistent with a saturation curve. In terestingly, the observed rates of excision showed some anomalies with his experiments too, with rates dropping for the 50 and 100nM AAG experiments and remaining more or less constant for the other single turnover experiments. Together our results may indicate an AAG-dependent change in the excision kinetics of A which needs further investigation to explain. Figure 6-5. Multiple to single tur nover titration of AAG with A T. 5nM substrate was used with 1.25 and 2.5nM AAG ( multiple turnover conditions ), 5nM AAG ( equal substrate and enzyme ) and 10, 20 and 40nM AAG ( single turnover conditions ). One turnover was seen at a fa ster rate under multiple turnover conditions ( See text and Table 6.3 )

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86 Table 6-3. Observed rates of multiple to si ngle turnover titration assays of AAG with 5nM A T AAG (nM) 1.25 2.5 5.00 10.0 20.0 40.0 kobs (min-1) 0.2 0.2 0.08 0.1 0.1 0.1 Product (nM) 1.0 1.8 2.60 0.4 0.6 1.2 Optimization of Assay Conditions for Maximum AAG Activity The loss of AAG activity at 37 C may be due to nonoptimum assay conditions, which may destabilize AAG especially at hi gher temperatures. Variations in buffer composition including pH, salts and other st abilizing agents like BSA may improve stability. A recent publication has suggested that for neutral substrates like Hx and A, pH optimum for efficient excision by AAG is 6.0. The pH optimum is 6.0 for neutral substrates to make them favor able leaving groups upon excision ( 109 ). The assay buffer that we routinely use was based on previous da ta and stability assays and was at a pH of 8.0. To determine if excision was more effici ent at pH 6.0, assays were done at pH 6.0, with sodium acetate pH 6.0/sodium chlori de or potassium acetate pH 6.0/potassium chloride instead of HEPES pH 8.0/potassium chloride, which were the buffer and salt used in all our assays. Assays contained 50mM buffer and 100mM salt. The potassium buffer and salt were used at pH 6.0 because it was shown previously that potassium chloride was more suitable for storing a nd assaying AAG than sodium chloride. KCl stabilized AAG better than NaCl. An exci sion reaction was performed under these conditions with 5nM Hx T with G-C base stacking part ners and 50mM AAG diluted in the respective storage buffer to maintain salt and pH conditions. The substrate was so chosen that any increase or decrease in activ ity will be very evident, this being the

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87 slowest excised substrate of all and probably the most stable substrate. The observations were interesting though by no means reflectiv e of AAG activity being improved at pH 6.0. Only the potassium acetate pH6.0/potassium chloride buffer showed more products formed. The sodium acetate pH 6.0/sodium chloride buffer showed activity similar to the HEPES pH 8.0/potassium chloride, which was the previously used buffer/salt combination (Figure 6-6). The rate of pr oduct formation though remained essentially unchanged under all three conditi ons (Table 6-4). The argument that the right pH was not achieved to ensure maximum activity of AA G was not supported by these experiments. The effect of the potassium salts may be due to some other effect of salt on DNA structure and stability. To veri fy this, melting temp eratures were measured as described for the same substrate used in these assays in the respective buffers. The overwhelming observation was the lower Tm with Potassium acetate, pH 6.0/Potassium chloride. The Tm was lower by about 3 C than the Tm of 51.5 C seen with HEPES, pH8.0/Potassium chloride and Sodium acetate, pH 6.0/Sodium ch loride buffers (Table 6-4). Therefore, the effects of the potassium buffer may not be a pH effect but a salt effect on DNA stability or structure which may make Hx more flippable by AAG. The buffers used by the authors ( 113, 114 ) to illustrate the effects of lower pH on neutral substrates did not hold in the case of our assays since no enhanced excision of Hx was observed in the sodium buffer at pH 6.0 over our buffer at pH 8.0.

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88 Figure 6-6. Effect of pH and salt on excision of Hx T. 5nM Hx T with G-C stacking partners was used as substrate to assess the activity of 50nM AAG at pH 6.0 with either NaOAC/NaCl buffer or KOAc/KCl buffer. The activity was compared to the control excision assay with the same amount of AAG at pH 8.0 in HEPES/KCl, which are the usual conditions used by us. NaOAC/NaCl buffer at pH 6.0, which was reported to be the optimum buffer, did not improve AAG activity over our buffer. When Na was replaced by K at pH 6.0, enhanced activity was seen. Table 6.4. Comparison of the effects of pH and salt on AAG activity and stability with 5nM GHxC T Buffer Product formed (nM) kobs (min-1) Tm ( C) HEPES/KCl, pH 8.0 1.4 0.1 51.5 NaOAc/NaCl, pH 6.0 1.8 0.1 51.5 KOAc/KCl, pH 6.0 2.9 0.05 47.5 Being convinced that the pH and salt in our assay buffer was in no way restricting enzyme activity, agents that were found to have stabilizing eff ects on proteins were considered. BSA is a common stabilizing agen t, used to store proteins and in assay buffers to prevent protein denaturation. BSA has been used by some groups in AAG buffers, but no extensive study on what effect s it had on AAG activity is available for

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89 reference. To determine if BSA would st abilize AAG, 1mg/mL and 0.1mg/mL BSA were used in AAG storage buffer to dilute the pr otein. Excision of 5nMT with the GC stacking partners was observed with 50nM AAG using th e standard AAG assay buffer at pH 8.0. A much less robust excision was noticed when compared to the N o BSA control, and lesser product was formed afte r 80 minute time course (Figure 6-7). Either BSA was not a good stabilizing agent or mo re concentration ranges of BSA must be tried before ascertaining its usefulne ss in stabilizing AAG. Figure 6-7. Effect of BSA on AAG stability and activity. Activ ity of 50nM AAG on 5nM Hx T with GC stacking partners was asse ssed in the presence of 0.1mg/mL and 1mg/mLBSA. Activity in the presence of BSA was reduced when compared to the No BSA control. Conclusions about Activity and Stability of AAG During Assays It was clear from the above experiment s that loss of activity at 37C was contributing to reduced AAG activity and this when combined with slow product release

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90 and product inhibition, could be the candida te issue preventing multiple turnovers by AAG. The issue of multiple turnovers due to instability, combined with poor substrate binding was seen to be exaggerated with slower substrates and not a factor with faster substrates. The reasons for loss of activity could be manifold, ranging from intrinsic instability of the protein to instability in th e particular c onditions used in the assays. Whatever be the reason, it is evident that a critical concentration exists, below which loss of activity is significant. Stab ilizing agents like BSA were not useful. The suggestion that pH optima decide the excision effici ency of different substrates ( 109,110 ) did not hold under these assay conditions, rather, a more general effect of salt on DNA was seen. The instability of AAG could be an artifact of the in vitro system used in these assays, due to the lack of other cellular f actors, including other BER prot eins, acting downstream of AAG or other factors not yet discovered. Or, the instability may ju st be a biological property of the protein which could be important in the cell. Additional experiments may give more detail s as to the best possible conditions for maximum activity and stability of AAG. The complexities associated with multiple turnover versus single turnover conditions ma y also begin to become more clear with experiments addressing the issue of stability at various conditions.

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91 CHAPTER 7 FLUORESCENCE ASSAYS TO OBSE RVE BINDING AND EXCISION OF ETHENOADENINE BY AA G IN REAL TIME The need to develop an experiment which would allow us to look at base flipping and excision by AAG in real time is great given the results obtained from the intercalating mutants and the sequence contex t effects. The most probable experiment would be to exploit the natu ral fluorescence properties of A and use its changes to observe A flipping and excision by AAG. This experi ment will be valuable in validating our interpretations of the effect of the Ty r-162 mutations on flipping and the effects of base stacking neighbors. The difluorotoluene complement will also serve as another tool to test these conclusions. Real time measur ements can also give equilibrium binding constants and a means to measure the rate of flipping, which can alleviate the limitations of the EMSA, which was the assay used to obs erve binding of AAG to its substrates. The crystal structure suggests that the fluorescence of A has a potential to change when it is flipped out of the base stack into the activ e site and then rele ased into solution upon excision of the glycosylic bond. There is also the possibility that the rate and magnitude of these changes is different and with the co mbination of the catalytically inactive E125Q mutant and the active AAG 79, flipping and excision can be monitored independent of each other to complement the 32P assays for excision and binding respectively. A is naturally fluorescent, with an max of excitation at 310nm and a max of emission at 405nm( 94 ). Its fluorescence is quite que nched when stacked in the DNA

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92 double helix. But upon binding and excision by AAG, it shows an increase in intrinsic fluorescence as observed in the excitati on and emission spectra (Figure 3-2) Fluorescence of A During Binding by AAG In this experiment, A fluorescence in duplex DNA was monitored at room temperature with saturating concentrati ons of E125Q (400-1600nM). These conditions mimicked the EMSA conditions, in which bi nding was observed independent of excision. The goal of the E125Q experiment was to see whether the flip ping step could be observed and its rates measured. Hand-mi xing experiments in which AAG was added to A-DNA were done in the fluor imeter. Fluorescence of A in DNA was largely quenched due to base stacking, so when A is flipped out by AAG, a fluorescence intensity increase can help monitor the fli pping step. No increase in fluorescence intensity of A-DNA only was observed. Immediately following addition of enzyme to the DNA in the cuvette, there was a sharp increase in fluores cence intensity of A and then constant fluorescence at 410nm. This jump in fluorescence could indicate a very fast A flipping step, since the intensity was higher than that of stacked A in DNA. But the increase was very fast and in the time scale us ed in this instrument, it was not possible to monitor the real change in fluorescence over time, as planned. Alternately, the increase in fluorescence could be due to Trp fluorescen ce when AAG was added to DNA. Titration experiments were done with 100nM duplex DNA containing A and 25 to 800nM E125Q, which is the general scale used for EMSA experiments. There was a progressive increase in the intensity of A fluorescence with increasing E125Q with the signal saturating with 400 and 800nM E125Q (Figure 7-1). If the increas e in fluorescence was due to the addition of AAG, then the signal must not saturate but keep on increasing with increasing enzyme. The saturated signal and previous observations from the EMSA made

PAGE 106

93 us interpret the fluorescence increase as a re flection of the rapid binding and flipping of A. Hence, this jump in A fluorescence intensity could be indicative of the flipped AE125Q complex. Interestingly, the magnitude of the fluorescence increase was the same for A in any sequence context (T A G substrates or G A C substrates) (Figure 7-1). It is important to recall that neither the base pairing partner nor base stacking neighbors changed the binding and excision of A by AAG ( 75 ). A does not hydrogen bond to any other base and is bulkier than the other AAG substrate studied in this work Hx. This similarity in the fluorescence intensity changes of A in both sequence contexts complements the EMSA, which showed very little change in the fraction bound for both DNA substrates. Figure 7-1. 500 second time based fluorescence of A when bound by increasing concentrations of E125Q 100nM DNA with A in two sequence contexts, T A G (left panel) and G A C (right panel) was used. Excitation was at 320nM and fluorescence emission was observed at 410nM with the band pass set at 4nM. The DNA only background was recorded for 100 seconds after which the shutter was opened and the enzyme added. A sharp jump in fluorescence emission upon closing the shutter wa s observed, followed by constant fluorescence. The sequence context did not affect the change ( See text )

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94 Blueshift of A Emission upon Binding by E125Q Upon titrating increasing concentrations of E125Q with DNA substrates containing A as the damaged base, as discussed above, emission spectra were taken to observe the max of emission when excited at 310-320nm. W ith increasing E125Q concentrations, a blue shift occurred in the emission maximum from 410nm, which is the A emission max to 390nm with 800nM E125Q with the shift being gradual with increasing enzyme (Figure 7-2). This shift was similar with both A substrates used and the progress of the blue shift coincided with the increase in fluorescence intensity observed over time in Figure 7-1. Interestingly, a co rresponding spectrum of increas ing concentrations of the enzyme only did not show any emission maxi ma or blue shift when excited at 320nm (Figure 7-3). This control was incorporated to rule out any possibl e fluorescence change in the enzyme, as, it is possible that at th e UV range, protein fluor escence is significant and may affect the observed signal. Figure 7-2. Emission spectra of 100nM A substrates, after adding 25-800nM E125Q at room temperature, after excitation at 320nm. A was placed in two sequence contexts, T A G ( left panel) and G A C ( right panel). The red arrow indicates the normal emission maximum of A at 410nm, as can be seen with the free DNA spectrum. The blue arrow indicates the shifted max with increasing E125Q.

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95 Figure 7-3. Emission scans of increasing concen trations of E125Q only when excited at 320nm. No appreciable fluorescence of the protein was observed and no blue shift was observed, contrary to when e125Q bound ADNA ( See Figure 7.2 and text ) The Y162S mutant, which did not show a shifted band with A and Hx containing DNA in the mobility shift assays (Chapter 4) did not show any a ppreciable increase in fluorescence with A. The Y162S mutant did not blue shift with increasing concentrations of enzyme when excited at 320nm at room temperature (Figure 7-4). The blue shift with E125Q was interpreted as a po ssible electrostatic change in the active site of E125Q upon flipping the A base out of the DNA. Hence the gradual blue shift with increasing enzymeflipped A complex formation. The electrostatic change upon flipping would also explain the lack of the shift with the en zyme only and with a flippingdeficient mutant, Y162S. Again, the Y162S e xperiment proves that this mutant was deficient in flipping the damaged base, and Tyr-162 is responsible largely for bringing and stabilizing the enzyme -flipped base complex.

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96 Figure 7-4. Emission spectra of 100nM TEG with 100, 200 and 800nM Y162S. The Y162S only, DNA only and buffer only cont rol spectra are also shown to bring out the lack of the blue shift ob served with E125Q. Th ere is neither an increased fluorescence emission from A nor a blue shift accompanying the increase. Fluorescence of A upon Excision of A by AAG Experiments were also done under excisi on conditions to observe the change in fluorescence when A was excised by AAG. Excision of A may give a much higher change in signal than that observed with E125Q mediated binding. If the excision reaction can be monitored by observing A fluorescence change and found to complement the 32P-based gel assays, we could have another realtime technique which can give us many more time points th an the gelbased excision assays. When AAG 79 was added to the cuvette cont aining DNA and the fluorescence of A recorded at 37 C, an increase in fluorescence of A was observed when excited at 310nm with an emission maximum at 420n m. After observing the background fluorescence of 100nMDNA only, 400 or 800nM enzy me were added and the shutter was closed. A sharp jump in A fluorescence was observed. But in contrast to E125Q binding,

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97 after the sharp jump, a slow and gradual increase in fluorescence intensity over a long time, up to 60 minutes was observed, when th e enzyme was capable of excision (Figure 7-5). The progress of the fluorescence increase matched the progress of a A excision curve obtained from 32P-based excision assays. The magnitude of the first jump in fluorescence was also smaller than the incr ease seen with E125Q mediated binding. The jump is interpreted as binding and flipping of A into the active site, whereas the slower, timebased increase is the release of free A after excision by AAG. Interestingly the magnitude of the initial jump was higher fo r E125Q than for wtAAG. The difference in the initial increase was interpreted for now as possibly arising from the mutation which removes the charged glutamate and replaces it with neutral glutamine. The loss of glutamate could increase the fluorescence of the base-flipped complex. In contrast to binding only, excision of A by AAG did not cause a blue shift the in the excision max (Figure 3-2).

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98 Figure 7-5. Timebased increase in A fluorescence upon excision by 800nM AAG at 37 C ( green trace ). 100nM T AG was incubated at 37 C and excited at 310nm and its emission observe d at 420nm. After 300 sec onds (flat part of the curve) 800nM AAG was added and the shut ter closed. A sharp increase in A fluorescence was observed, followed by a gradual time-based increase in fluorescence indicative of excision of A. The progress of the curve is reminiscent of a curve obtained by fitting 32P-based excision assays. The contrast between fluorescence change during excision and change during binding only is highlighted by the blue trace which represents binding by E125Q (see text). Stopped-flow Analysis of A Flipping by AAG It was clear from the experiment detaile d above and the observation of Figure 7-4, that even if the first jump in fluorescence was indicating flipping of A, it was too fast to allow any kinetic measurements of flipping. To directly measure the rate of this rapid increase in fluorescence, a stopped-flow analysis of A flipping was done, so that the changes associated with A flipping can be monitored on a millisecond time-scale. After measuring initial signals with free A and 100 and 200nM A T substrate, 200nM

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99 substrate was chosen for binding experiments because it provided a relatively intense signal. From EMSAs shown in chapter 4 and 5, it was noted that good binding of AAG to A substrates was seen with as little as 4-fold less enzyme than substrate. To rule out the possibility of binding of A by AAG being too rapid as is the case with handmaxing experiments, the stopped-flow was ma intained at a temperature of 20 C, at which the binding reaction is slower. The binding assay was done by rapidly mixing to give a final concentration of 100nM AAG and 200nM A substrate as described in Chapter 3. The change in fluorescence was monitored using 385nm cuton filters and a 5 milliseconds dwell time to collect data point s. After trials with 4-8 fold more enzyme than substrate, the fluorescence signal upon ra pid mixing was seen to be saturated. A gradual timebased change was not seen. This rapid cha nge could either mean that the fluorescence change was not indicative of flipping or that the binding reac tion is too fast that the signal is saturated at the onset. So, in an attempt to slow down the binding reaction, lower enzyme concentrations at which saturated binding may not be seen were used. After referring back to a previous EMSA, 100nM AA G was chosen as a concentration at which binding could be slower due to the fact that saturation of the subs trate by E125Q was not observed at this concentration in the EMSA. With 100nM AAG and 200nM A T substrate, a gradual increase in fluorescence, spanning the first 10 se conds of the binding reactions was observed (Figur e 7-6). The signal change wa s very small in magnitude when normalized with the fluorescence signa ls associated with the AAG only and the DNA only controls. The signal: noi se ratio was also very small. These two factors made any kinetic interpretation of the fluorescent increase highly error-prone. Nevertheless,

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100 this could be the beginning of a real-time experiment to measur e the rate of baseflipping. Figure 7-6. Stopped-fl ow analysis of A flipping by AAG. 200nM A T and 100nM AAG were mixed in the stopped-flow machine at 20 C and the fluorescence change associated with binding was m onitored after excitation at 310nm using 385nm cut-on filters. A gradual change in A fluorescence was noticed very early in the reaction. Contribution of Fluorescence Experi ments to Understanding of AAG The need for a real time assay for glycosyl ases cannot be overstated, especially for AAG for which base flipping makes an im portant contribution to activity. The availability of the natura lly fluorescent substrate, A for development of a real time assay was also very fortunate. In the first group of experiments described in this chapter, it was clear that A fluorescence changes when flipped and excised by AAG. The Y162 mutants whose properties were analyzed and detailed in Chapter 4 are valuable reagents to use with A. The fluorescent base and flipping mutant s, when combined tell us more about

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101 the contribution of flipping to the activity and diversity of AAG, which is the primary objective of this dissertation. The EMSA which was the binding assay used quite successfully to delineate the differences between the Y162 mutants on one hand and the sequence context effects on the other hand, is not an equilibrium technique. So, the development of an assay to determine rates of flipping in real time would be very useful in ascertaining the conclusions made in Ch apters 4 and 5. To this end, the fluorescent assays looking at the change s in the emission spectra and A intensity upon binding by AAG are significant. The blue shift observed upon binding and the real time increase in fluorescence observed in the stoppedflow anal ysis are important indications that with more fine tuning, a real time assay to measure flipping rates is possible. That is the most important conclusion made from the e xperiments reported in this chapter.

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102 CHAPTER 8 DISCUSSION AND FUTURE DIRECTIONS This dissertation has attemp ted to address the questi on of the extent of the contribution of flipping to the overall e fficiency of DNA gl ycosylases by studying flipping in AAG. AAG is the idea l glycosylase to use in such a study owing to its diverse substrate range and the availability of structural data to plan an effective strategy to test our hypotheses. The expectati on that the contribution of base flipping could be considerable to AAGs activity was vindicated by the results obtain ed from mutagenesis of both the protein and the DNA. But how can these results be comb ined to explain the effects of flipping efficiency on the over all activity of the pr otein based on our preliminary twostep model for AAG activity? The two-step model proposed, served as a basis for partitioning the ac tivity of AAG outside the activ e site (such as substrate recognition, DNA binding and ba se flipping) and the act ual catalysis activity accomplished by the active site ( 75 ). But this partitioning is strictly theoretical, a good first step to understand two comp lex activities, infinitely useful in formulating strategies to look at flipping effects. The truth is that, both steps are interdepe ndent and affect each other, reflecting on the activity of the enzyme. If ever the purpose of this dissertation can be achieved, it would be by formulating a model that can explain this inevitable connection between the two steps. A good way to do that would be to revisit the basic two-step model and incorporate the kineti c interpretations detailed in the preceding chapters. The purpose of this discussion is to accomplish the goal to applying the kinetic

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103 conclusions made in chapters 4 and 5 to come up with a better understanding of the mechanism of AAG activity and substrate diversity. The Night-Watchman Model for AAG Activity The Night-watchman model for AAG activity is a comprehensive twostep model (Figure 8-1). According to this model, AAG can scan along the minor groove randomly looking for bases that are susceptible to f lipping. It must be recalled here that AAG substrates like 3-MeA, Hx and A have different properties in DNA from the normal bases, and hence will be flippable during th is process, while normal bases will not. Once flipped, various factors in the active site of AAG including proper f it, alignment of the nucleotide for excision and suitability of the leaving group, all play a role in deciding the efficiency by which base excision proceeds to complete AAG activity. This second step is an effective check for any accidentally flipped normal bases. A general acidbase mechanism of excision serves to accommodate diverse substr ates. This whole process is akin to how a nightwatchman checks for open doors along an alley and, when he does encounter an open door, checks for any possibl e problems associated with the open door. Hence, the name Nightwatchman mode l for AAG activity is an apt coinage. This model can delineate the 2 steps enough to test them separately but at the same time allows for a common mechanism in which one factor can affect both steps in tandem and hence affect both the substrate recogni tion and excision of the damaged base by AAG. This model is also very useful in pr opagating the mechanism of AAG to include other possible steps like product dissociation and recycling back to the substrate, and this possible addition is a very important futu re goal in understanding AAG mechanism and action.

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104 Figure 8-1. The Night-watchman model for AAG activity. AAG scans the minor groove for possible substrates by checking first for flippabilty and second for excisability once flipped into the ac tive site. This twostep mechanism provides for enough leeway to act on multip le substrates and at the same time control over excising normal bases and nonsubstrates. There are many variables that can affect St ep1. This thesis has examined two such variables that could poten tially affect Step1-Tyr-162, th e AAG residue responsible for intercalating into the minor groove and enabling the flippe dout conformation of the AAGDNAflip complex and local sequence context wh ich can impede or facilitate the achievement of the AAGDNAflip complex Tyr-162 and the Flipping Equilibrium As seen in figure 8-1, the flipping equilibrium can affect the active sitemediated catalysis by making less substrate availabl e for excision by AAG. Tyr-162 is the AAG residue responsible for bringi ng about flipping and stabilizing the resultant flipped out baseAAG complex to enable excision. This complex function is accomplished by Tyr162s ability to intercalate into the DNA and occupy the space vacated by the damaged base. When compared to the binding mechanis ms of other wellstudied glycosylases like AAG + DNA AAG-DNA AAG-DNA fli pBASE EXCISION STEP 1 CHECK FOR FLIPPABILITY STEP 2 CHECK FOR EXCISABILITY

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105 UDG, which uses a pinch-push-pull mechanism ( 85 ) to flip out uracil from DNA, the crystal structure of AAG bound to A did not reveal any such distorting mechanisms for flipping A. The contribution of Tyr-162 mediated intercalation, therefore, was thought to be very important in flipping the dama ged base. This model was tested by mutating Tyr-162 to Phe and Ser. The Y162S mutant had undetectable ac tivity, whereas, the Y162F mutant had reduced activity. In EMSAs, it was seen that binding affinity was much reduced for the Y162F mutant with both Hx and A substrates. This reduced activity can be attributed to impaired intercalation due to the mutation. Ser, lacking the aromatic residue of Tyr was severely impair ed, while Phe lacking the hydroxyl group of Tyr was significantly impaired. This impair ment can reflect on Step-1 of the Nightwatchman model, by making flipping inefficien t, and thus move the equilibrium to the left, leaving less substrate f lipped and hence less substrat e excised (Figure 8-2). The combined effect of the mutation would be less efficient binding and slower rates of excision, both resulting from less efficient flipping as a result of the mutation. Figure 8-2. Compromised flippi ng due to Tyr-162 mutation. Reduced intercalation ability in the Y162F mutant can reflect on the flipping equilibrium and result in compromised flipping. Less Y162F-flippe d base complex formation makes excision rates lower owing to less substrate availability in the active site. The effect of the mutation was overcome by removal of hydrogen bonds to Hx, hence making it less stable in DNA and more easily flipped Y162F + DNA Y162F-DNA Reduced Y162F-DNA fli p Lower rates of Excision STEP 1 COMPROMISED FLIPPING STEP 2 LESS SUBSTRATE AVAILABLE

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106 Stability of Hx and the Flipping Equilibrium The activity of the Y162F mutant was rescue d when Hx was base paired with F, the reason being that Hx cannot form hydrogen bonds with F. The additional energy needed to break the hydrogen bonds and flip out the base may also be primarily provided by the stabilizing effect of Tyr-162 intercalation into DNA. In the absence of Tyr-162, the less stable a substrate is in DNA, the more likely it is to be flipped out by the mutant. The reduced stability of Hx in a nonhydrogen bonded Hx F context compared to the wobble basepaired Hx T context can thus, move the flippi ng equilibrium in opposite directions, reflected in enhanced or slower rates of excision. The success with the Tyr-162 mutants and th e effects of base pairing partners on Hx excision prompted to look at other mean s to perturb the flip ping equilibrium. This time around, Hx was placed in either the stronge st or the weakest base stacking context and base paired with T, F or C. The resulting single turnover excision rates reflect the flipping equilibrium with no change in the intrinsic rate of chemistr y in any way. The flip ping equilibrium, moved either way based on the sequence context ar ound Hx. This is attested by the EMSAs shown in Chapter 5, in which the intens ity of the higher molecular weight band, indicative of a flipped base-AAG complex, is inversely proportional to the stability of Hx. More bound Hx is seen at a lower AAG concentration when in a weaker base stacking/ non-hydrogen bonding co ntext. In a stronger base -stacking/ hydrogen bonding context, less Hx is bound and more AAG is re quired to bind that Hx. This property is reflected in the amount of AAG required to br ing about saturated ex cision of Hx in the respective sequences. That these sequence changes could intrinsically affect DNA

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107 stability was also verified by measuring melting temperatures which also complemented the excision and binding results. So, for Hx, perturbing the flipping equilibrium changed the way it is perceived as a substrate by AAG. The stability of Hx made it either a better or wors e substrate solely by affecting how well it is flipped by AAG. This may mean that, for Hx, the ratelimiting step in its excision by AAG could be fli pping, as illustrated in Figure 8-3. The hydrogen bonding potential of Hx to T and C also suppor ts this concept. A rate-limiting flipping step for Hx can have a biological significan ce owing to the fact that, flipping decides whether Hx is a substrate for excision or not, and flipping Hx T and Hx C will have totally different consequences. Hx T flipped, relatively well by AAG must be excised before an additional round of replication to reverse the promutagenic effects of Hx. On the other hand, Hx C is highly mutagenic. Flipping Hx C and thereby excising Hx may confirm an AT GC transition even before a round of replication. Hence, there seems to be a biologic imperative to control flipping of Hx by AAG. The base stacking effects add another layer to this story. and the combination of sequence effects may decide the occurrence of mutations in pa rticular sequences of the ge nome, adding leverage to the existence of possible mutational hot-spots ve rsus cold-spots, which are regions of the genome prone to having mutations versus mutationfree regions. The difference may be as simple as slower repair of lesions versus faster repair of lesions as is possible based on the sequence context a lesion like Hx is found. The objective of th is dissertation of studying the contribution of flipping to the ac tivity of AAG has thus been completed to its limits in coming up with this interpretation.

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108 Figure 8-3. The effect of Hx stability on the Night-watchman model. Stability of Hx in DNA is affected by the sequence context of Hx which in turn affects the flipping equilibrium in Step 1. A rate-limiting flipping step would mean that enhanced or compromised flipping w ould increase or decrease rates of excision without any intrinsic changes in the actual rate of chemistry Not All Bases Are Born Equal Another question that pops up from the resu lts obtained in Chapters 4, 5 and 6 is, what about A? What makes A practically immune to the se quence context effects that so dramatically affected Hx? The answer lies in the fact that the two bases are not the same. A, in contrast to Hx, does not form hydr ogen bonds in DNA and is a bulky base. The stability of A is not likely to be compromised by sequence changes around the base. Progressive degradation of A leads to many intermediates which can be mutagenic ( 74 ). The lack of hydrogen bonding in DNA and bulky nature combine to make A a much more distorting lesion in DNA and confer the possi bility of it being pa rtially extra helical. This property may enable easy flipping by AAG. Binding assays using the EMSA show that A is bound very efficiently, with low na nomolar binding constants, and needs only twice as much enzyme than substrate to bi nd all. This is cont rasting to the binding properties of Hx. The efficien t binding in the EMSA is i ndicative of very efficient flipping of A by AAG. What these assays indicate is that the flipping equilibrium is AAG + Hx DNA AAGHx -DNA AAGHx -DNA fli p STEP 1 RATE LIMITING FLIPPING STEP 2 EFFICIENT EXCISION INCREASED STABILITY DECREASED STABILITY Hx excisio n

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109 moved to the far right with A, leaving about 90% substrate flipped at a concentration of AAG at which only 10% Hx will be flipped. So sequence changes lik e base stacking and base pairing partners do not have the same effect on A as they have on Hx, since no hydrogen bonds need to be broken to flip A and the flipping equilibr ium is always tilted to the right. It is clear then that, unlike for Hx, flipping is not the ratelimiting step in A excision. Figure 8-4. Effect of A excision on the Night-watchman mo del. Rate limiting chemistry would follow efficient flipping of A to lead to compromised excision in the active site due to many factors. Then what else is likely to limit A excision? Single turnover excision of A have always shown almost invariant rates of exci sion with increasing AAG, with those rates being almost 4-times slower than the ra tes of Hx excision. The bulky nature of A may make chemistry in the active site compromised due to many factors, like a proper fit in the active site, alignment of the glycosid ic bond etc. So, it is likely that for A, chemistry is rate-limiting and not flipping. So, Step 2 of the Night-watchman model is limiting for A while Step 1 is limiting for Hx as is illu strated in Figure 8.4. This kinetic partitioning may be an important contribu tor to the ability of AAG to excise structurally diverse substrates. AAG + A-DNA AAGA-DNA AAGA-DNA fli p Base excision STEP 1 EFFICIENT FLIPPING STEP 2 RATE LIMITING CHEMISTRYFIT IN THE ACTIVE SITE, ALIGNMENT FOR EXCISION, ETC.

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110 Future Directions The most important out come of this disse rtation has been an understanding of the underlying complexity of DNA glycosylase ac tivity, especially owing to AAGs diverse substrate range. The use of novel and appropr iate methods to further the understanding afforded by the kinetic data discussed is im portant for future proj ects. The following are some suggestions as to what direct ions further research on AAG can take. Completing the Need for a Real Time Assay to Measure Flipping A most important corroboration to the conc lusions made in this chapter would be real time rates of base flipping by AAG. A di rect measure of the ease of flipping would confirm the model postulated and have a tremendous impact in the DNA glycosylase field, where the flipping mechanism is a majo r interest. An attempt towards developing such a technique was made and has been de tailed in chapter 7. But as concluded in chapter 7, further experiments and conditions ne ed to be worked out before the data can be kinetically interpreted to give flipping rates, which were beyond the scope of this dissertation. So, further trials w ith the stopped-flow fluorescence of A are essential. It can be argued that, only for Hx will such a technique be useful in corroborating the model since A was not affected by possible change s in the flipping equilibrium. An important avenue of research must be ways to report on Hx flipping by AAG. Fluorescent bases such as 2-aminopurine are available, wh ich can be incorporated into DNA in such a way that, their fluorescence intensity can change with the conformation of the neighboring base, say Hx. Such an experiment will be an enormous step forward in realizing the goal of a realtime assay for f lipping rates. With all the reagents made available by the projects undertaken for this dissertation, once such an experiment is established, the volume of informa tion obtainable is very large.

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111 Continuing Research on Sequence Context Effects The dramatic effect of local sequen ce context on Hx excision and not on A excision, combined with the rescue of the Y 162F mutant, told an important story about the preferences and fine-tuning of AAGs multiple substrate specificity. To continue the research started in this dissertation to include more sequence changes, not just to flanking neighbors but to more extensive neighbor ing bases will not only add on to the conclusions made, but also be relevant in the sense of the genome. Another weapon in this arsenal, is the availabili ty of the nonnative base, zebu larine (Z), which is a thymine analog that can form only one hydrogen bond with Hx (Figure 8-5). The use of zebularine as a base pairing partner can help to touc h the middle ground between T, with which Hx forms two wobble base pairs, C, with which Hx forms two WatsonCrick base pairs and F, with which Hx forms no base pairs. The outcome of these experiments could be very interesting and may ultimately provide the ground work to assess the effects of higher order DNA structures like chromatin and methylation on AAG activity. Figure 8-5. A hypoxanthine zebularine base pair. Hx can form one hydrogen bond with Z in contrast to two hydrogen bonds with T and C and none with F. Possible Strategy to Explain A Excision The only conclusion about the fate of A which is only excised by AAG was that, perturbing the flipping equilibrium did not affect its excision by AAG. The additional indication from the kinetic stud ies indicated that, chemistry of bond cleavage may be the most important step in A excision. The bulky nature of A, compared to the other AAG

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112 substrates, due to the presence of the etheno adduct on adenine, coul d be making it fit less productively in the active site for fast chem istry. This hypothesis can be tested by making the base binding pocket more amenable to f it a bulky base. The crystal structure of AAG bound to A shows an active site residue, Met169 making contacts with the etheno bridge of A (Figure 8-6). The contact may be the f actor limiting a proper fit in the active site thereby limiting chemistry. Mutating Met-169 to a smaller residue like alanine may make a proper fit more possible for A. Single turnover excision a ssays can then de done to compare the properties of this mutant on wild type AAG to see if A excision is improved in anyway. Binding controls by way of EMSAs need to be done to ascertain that the binding and flipping of A is the same for the mutant and the wild type. These experiments will be a way to propagate from analyzing Step 1 of the Nightwatchman model to analyzing step-2.The availa bility of two substrates, Hx and A is fortuitous in allowing both steps to be tested. The Met-169 mutant will also be an interesting way to look at Hx excision, since the binding pocket may allow movement of Hx in a way that could disrupt its alignment in the active site for chemistry. So, the Met-169 mutant will also add on to furthering the horizons of this dissertation.

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113 Figure 8-6. Crystal structure of the AAGA complex showing the proximity of Met-169 ( orange spacefill ) to the etheno bridge of the flipped out A ( yellow spacefill ) in the active site. The green sticks in the DNA Coordination of the Activity of AAG to Other Downstream Steps in BER It must be remembered that, though the DNA glycosylases perform the important function of ridding the genome of harmful dama ges, the product they leave behind is in fact, more harmful than their initial substrates. The abasic site which is the product of glycosylase activity is cyto toxic. In contrast to around half a dozen known human glycosylases, only one AP endonuclease, APE1, is known to process the abasic sites in humans. Moreover, spontaneous generation of ab asic sites proceeds at a considerable rate too, leading to around 10,000 abas ic sites/cell/day. Evidence is mounting on the possible coordination between glycosylases and endonucleases hOGG1 and APE1( 111 ), hMutY homolog and APE1( 112 ) as well as E. coli MutY and EndoIV and ExoIII( 113 ). A possible handover of product to successive enzymes in BER is now considered acceptable. The discovery of di rect proteinprotein intera ction between APE1 and Pol and between Pol and ligase III throu gh XRCC1 also add to th is concept. Numerous

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114 glycosylases and a single APE1 may not indicate direct protei nprotein interactions, but may have functional interactions that help to protect the cell from accumulating abasic sites. The single turnove r kinetics explored exte nsively in this dissert ation can be a very good place to start addressing th e possibility of functional interactions between AAG and APE1. The single turnover kinetics has explor ed the productindepe ndent part of AAG mechanism. The complex effects of product dissociation, substrat e re-association and enzyme inactivity need to be studied to fu rther the understanding of the pathway and look for functional interactions with downstream enzymes in the pathway. The observation that multiple turnovers by AAG were impaired can indicate a possi ble role for APE1, which is the next enzyme in the pathwa y, in relieving possible product inhibition on AAG. If a functional in teraction does exist, it may also be responsible for stabilizing AAG and may also relieve the loss of AAG ac tivity noticed at lower concentrations. A functional interaction between the first two enzymes of the BER pathway will strengthen the growing perception of passing the baton in BER It can be finally concluded that the ki netic analysis of AAG activity on Hx and A, undertaken in this dissertation has contributed significantly to the glycosylase field and will serve as the necessary ground work for mo re novel research in the critical field of base excision repair and the ultimat e goal of safe guarding the genome.

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115 LIST OF REFERENCES 1. Shapiro, R., and Klein, R. S. (1966) Biochemistry 5 2358-62. 2. Mosbaugh, D. W., and Bennett, S. E. (1994) Prog. Nucleic Acid Res. Mol. Biol. 48 315-70. 3. Lindahl, T. (1993) Nature 362 709-15. 4. Peng, W., and Shaw, B. R. (1996) Biochemistry 35 10172-81. 5. Marnett, L. J., and Plastaras, J. P. (2001) Trends Genet. 17 214-21. 6. Marnett, L. J., Riggins, J. N., and West, J. D. (2003) J. Clin. Invest. 111 583-93. 7. Atamna, H., Cheung, I., and Ames, B. N. (2000) Proc. Natl. Acad. Sci. U. S. A. 97 686-91. 8. Podlutsky, A. J., Dianova, II, Wilson, S. H ., Bohr, V. A., and Dianov, G. L. (2001) Biochemistry 40 809-13. 9. Gros, L., Saparbaev, M. K., and Laval, J. (2002) Oncogene 21 8905-25. 10. Moncada, S., Palmer, R. M., and Higgs, E. A. (1991) Pharmacol. Rev. 43 109-42. 11. Prior, J. J., and Santi, D. V. (1984) J. Biol. Chem. 259 2429-34. 12. Burney, S., Caulfield, J. L., Niles, J. C., Wishnok, J. S., and Tannenbaum, S. R. (1999) Mutat. Res. 424 37-49. 13. Caulfield, J. L., Wishnok, J. S ., and Tannenbaum, S. R. (1998) J. Biol. Chem. 273 12689-95. 14. Spencer, J. P., Whiteman, M., Jenner, A., and Halliwell, B. (2000) Free. Radic. Biol. Med. 28 1039-50. 15. Nilsen, H., and Krokan, H. E. (2001) Carcinogenesis 22 987-98. 16. Scharer, O. D., and Jiricny, J. (2001) Bioessays 23 270-81. 17. Memisoglu, A., and Samson, L. (2000) Mutat. Res. 451 39-51. 18. Lindahl, T. (2000) Mutat. Res. 462 129-35.

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116 19. Lindahl, T., and Wood, R. D. (1999) Science 286 1897-905. 20. Krokan, H. E., Nilsen, H., Skorpen, F., Otterlei, M., and Slupphaug, G. (2000) FEBS Lett. 476 73-7. 21. Sandigursky, M., Yacoub, A., Kelley, M. R., Xu, Y., Franklin, W. A., and Deutsch, W. A. (1997) Nucleic Acids Res. 25 4557-61. 22. Sobol, R. W., Prasad, R., Evenski, A., Ba ker, A., Yang, X. P., Horton, J. K., and Wilson, S. H. (2000) Nature 405 807-10. 23. Weiss, B. (1976) J. Biol. Chem. 251 1896-901. 24. Mol, C. D., Hosfield, D. J., and Tainer, J. A. (2000) Mutat. Res. 460 211-29. 25. Weiss, B. (2001) Mutat. Res. 461 301-9. 26. Chan, E., and Weiss, B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84 3189-93. 27. Demple, B., Herman, T., and Chen, D. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88 11450-4. 28. Robson, C. N., and Hickson, I. D. (1991) Nucleic Acids Res. 19 5519-23. 29. Osheroff, W. P., Beard, W. A., Wils on, S. H., and Kunkel, T. A. (1999) J. Biol. Chem. 274 20749-52. 30. Bhagwat, A. S., Sanderson, R. J., and Lindahl, T. (1999) Nucleic Acids Res. 27 4028-33. 31. Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81 7747-51. 32. Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V. A. (1999) J. Biol. Chem. 274 13741-3. 33. Fortini, P., Pascucci, B., Parlanti, E., Sobol R. W., Wilson, S. H., and Dogliotti, E. (1998) Biochemistry 37 3575-80. 34. Lieber, M. R. (1997) Bioessays 19 233-40. 35. Matsumoto, Y., Kim, K., and Bogenhagen, D. F. (1994) Mol. Cell. Biol. 14 618797. 36. Sobol, R. W., Horton, J. K., Kuhn, R., G u, H., Singhal, R. K., Prasad, R., Rajewsky, K., and Wilson, S. H. (1996) Nature 379 183-6. 37. Prasad, R., Dianov, G. L., Bohr, V. A., and Wilson, S. H. (2000) J. Biol. Chem. 275 4460-6.

PAGE 130

117 38. Prasad, R., Lavrik, O. I., Kim, S. J., Keda r, P., Yang, X. P., Vande Berg, B. J., and Wilson, S. H. (2001) J. Biol. Chem. 276 32411-4. 39. Sung, J. S., and Mosbaugh, D. W. (2003) Biochemistry 42 4613-25. 40. Bennett, R. A., Wilson, D. M., 3rd, Wong, D., and Demple, B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94 7166-9. 41. Kubota, Y., Nash, R. A., Klungland, A., Scha r, P., Barnes, D. E., and Lindahl, T. (1996) Embo. J. 15 6662-70. 42. Marintchev, A., Mullen, M. A., Maciejew ski, M. W., Pan, B., Gryk, M. R., and Mullen, G. P. (1999) Nat. Struct. Biol. 6 884-93. 43. Prasad, R., Singhal, R. K., Srivastava, D. K., Molina, J. T., Tomkinson, A. E., and Wilson, S. H. (1996) J. Biol. Chem. 271 16000-7. 44. Caldecott, K. W., Aoufouchi, S., Johnson, P., and Shall, S. (1996) Nucleic Acids Res. 24 4387-94. 45. de Murcia, G., and Menissier de Murcia, J. (1994) Trends Biochem. Sci. 19 172-6. 46. de Murcia, J. M., Niedergang, C., Trucco, C., Ricoul, M., Dutrillaux, B., Mark, M., Oliver, F. J., Masson, M., Dierich, A., LeMeur, M., Walztinger, C., Chambon, P., and de Murcia, G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94 7303-7. 47. Nilsen, H., Otterlei, M., Haug, T., Solum, K., Nagelhus, T. A., Skorpen, F., and Krokan, H. E. (1997) Nucleic Acids Res. 25 750-5. 48. Takao, M., Aburatani, H., Kobayashi, K., and Yasui, A. (1998) Nucleic Acids Res. 26 2917-22. 49. Ohtsubo, T., Nishioka, K., Imaiso, Y., Iwai S., Shimokawa, H., Oda, H., Fujiwara, T., and Nakabeppu, Y. (2000) Nucleic Acids Res. 28 1355-64. 50. de Souza-Pinto, N. C., Eide, L., Hogue, B. A., Thybo, T., Stevnsner, T., Seeberg, E., Klungland, A., and Bohr, V. A. (2001) Cancer Res. 61 5378-81. 51. Croteau, D. L., ap Rhys, C. M., Hudson, E. K., Dianov, G. L., Hansford, R. G., and Bohr, V. A. (1997) J. Biol. Chem. 272 27338-44. 52. Croteau, D. L., Stierum, R. H., and Bohr, V. A. (1999) Mutat. Res. 434 137-48. 53. Vaughan, P., Lindahl, T., and Sedgwick, B. (1993) Mutat. Res. 293 249-57. 54. Moore, M. H., Gulbis, J. M., Dodson, E. J., Demple, B., and Moody, P. C. (1994) Embo. J. 13 1495-501. 55. Lindahl, T. (1974) Proc. Natl. Acad. Sci. U. S. A. 71 3649-53.

PAGE 131

118 56. Matijasevic, Z., Sekiguchi, M ., and Ludlum, D. B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89 9331-4. 57. Bjelland, S., Bjoras, M., and Seeberg, E. (1993) Nucleic Acids Res. 21 2045-9. 58. Riazuddin, S., and Lindahl, T. (1978) Biochemistry 17 2110-8. 59. Thomas, L., Yang, C. H., and Goldthwait, D. A. (1982) Biochemistry 21 1162-9. 60. Samson, L., Derfler, B., Boosalis, M., and Call, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88 9127-31. 61. Chakravarti, D., Ibeanu, G. C., Tano, K., and Mitra, S. (1991) J. Biol. Chem. 266 15710-5. 62. O'Connor, T. R., and Laval, J. (1991) Biochem. Biophys. Res. Commun. 176 11707. 63. Vickers, M. A., Vyas, P., Harris, P. C ., Simmons, D. L., and Higgs, D. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90 3437-41. 64. Pendlebury, A., Frayling, I. M., Santiban ez Koref, M. F., Margison, G. P., and Rafferty, J. A. (1994) Carcinogenesis 15 2957-60. 65. Bouziane, M., Miao, F., Bates, S. E ., Somsouk, L., Sang, B. C., Denissenko, M., and O'Connor, T. R. (2000) Mutat. Res. 461 15-29. 66. Engelward, B. P., Weeda, G., Wyatt, M. D ., Broekhof, J. L., de Wit, J., Donker, I., Allan, J. M., Gold, B., Hoeijmakers, J. H., and Samson, L. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94 13087-92. 67. Elder, R. H., Jansen, J. G., Weeks, R. J., Willington, M. A., Deans, B., Watson, A. J., Mynett, K. J., Bailey, J. A., Cooper D. P., Rafferty, J. A., Heeran, M. C., Wijnhoven, S. W., van Zeeland, A. A., and Margison, G. P. (1998) Mol. Cell. Biol. 18 5828-37. 68. Miao, F., Bouziane, M., Dammann, R., Masu tani, C., Hanaoka, F., Pfeifer, G., and O'Connor, T. R. (2000) J. Biol. Chem. 275 28433-8. 69. Asaeda, A., Ide, H., Asagoshi, K., Matsuyama, S., Tano, K., Murakami, A., Takamori, Y., and Kubo, K. (2000) Biochemistry 39 1959-65. 70. Dosanjh, M. K., Chenna, A., Kim, E., Fr aenkel-Conrat, H., Samson, L., and Singer, B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91 1024-8. 71. Dosanjh, M. K., Roy, R., Mitra, S., and Singer, B. (1994) Biochemistry 33 1624-8. 72. van der Kemp, P. A., Thomas, D., Barbe y, R., de Oliveira, R., and Boiteux, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93 5197-202.

PAGE 132

119 73. Leithauser, M. T., Liem, A., Stewart, B. C ., Miller, E. C., and Miller, J. A. (1990) Carcinogenesis 11 463-73. 74. el Ghissassi, F., Barbin, A., Na ir, J., and Bartsch, H. (1995) Chem. Res. Toxicol. 8 278-83. 75. Vallur, A. C., Feller, J. A., Abner, C. W., Tran, R. K., and Bloom, L. B. (2002) J. Biol. Chem. 277 31673-8. 76. Abner, C. W., Lau, A. Y., Ellenberg er, T., and Bloom, L. B. (2001) J. Biol. Chem. 276 13379-87. 77. Lau, A. Y., Scharer, O. D., Samson, L., Ve rdine, G. L., and Ellenberger, T. (1998) Cell 95 249-58. 78. Lau, A. Y., Wyatt, M. D., Glassner, B. J., Samson, L. D., and Ellenberger, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97 13573-8. 79. Klimasauskas, S., Kumar, S., Roberts, R. J., and Cheng, X. (1994) Cell 76 357-69. 80. Reinisch, K. M., Chen, L., Verdine, G. L., and Lipscomb, W. N. (1995) Cell 82 143-53. 81. Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, H. E., and Tainer, J. A. (1996) Nature 384 87-92. 82. Yamagata, Y., Kato, M., Odawara, K., Tokuno, Y., Nakashima, Y., Matsushima, N., Yasumura, K., Tomita, K., Ihara, K ., Fujii, Y., Nakabeppu, Y., Sekiguchi, M., and Fujii, S. (1996) Cell 86 311-9. 83. Barrett, T. E., Savva, R., Panayotou, G., Barlow, T., Brown, T., Jiricny, J., and Pearl, L. H. (1998) Cell 92 117-29. 84. Fuxreiter, M., Luo, N., Jedlovszky, P., Simon, I., and Osman, R. (2002) J. Mol. Biol. 323 823-34. 85. Parikh, S. S., Mol, C. D., Slupphaug, G., Bhar ati, S., Krokan, H. E., and Tainer, J. A. (1998) Embo. J. 17 5214-26. 86. Fraser, C. M., Casjens, S., Huang, W. M ., Sutton, G. G., Clayton, R., Lathigra, R., White, O., Ketchum, K. A., Dodson, R., Hickey, E. K., Gwinn, M., Dougherty, B., Tomb, J. F., Fleischmann, R. D., Richardson, D., Peters on, J., Kerlavage, A. R., Quackenbush, J., Salzberg, S., Hanson, M., van Vugt, R., Palmer, N., Adams, M. D., Gocayne, J., and Venter, J. C. (1997) Nature 390 580-6. 87. Morohoshi, F., Hayashi, K., and Munkata, N. (1993) J. Bacteriol. 175 6010-7. 88. Santerre, A., and Britt, A. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91 2240-4.

PAGE 133

120 89. Cole, S. T., Brosch, R., Parkhill, J., Garn ier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., 3rd, Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Horn sby, T., Jagels, K., and Barrell, B. G. (1998) Nature 393 537-44. 90. Labahn, J., Scharer, O. D., Long, A., E zaz-Nikpay, K., Verdine, G. L., and Ellenberger, T. E. (1996) Cell 86 321-9. 91. Handa, P., Roy, S., and Varshney, U. (2001) J. Biol. Chem. 276 17324-31. 92. Jiang, Y. L., and Stivers, J. T. (2002) Biochemistry 41 11236-47. 93. Jiang, Y. L., Stivers, J. T., and Song, F. (2002) Biochemistry 41 11248-54. 94. Secrist, J. A., 3rd, Barrio, J. R., Leonard, N. J., and Weber, G. (1972) Biochemistry 11 3499-506. 95. Stivers, J. T., Pankiewicz, K. W., and Watanabe, K. A. (1999) Biochemistry 38 952-963. 96. Savva, R., McAuley-Hecht, K., Brown, T., and Pearl, L. (1995) Nature 373 487493. 97. Mol, C. D., Arvai, A. S., Slupphaug, G., Ka vli, B., Alseth, I., Krokan, H. E., and Tainer, J. A. (1995) Cell 80 869-878. 98. Jiang, Y. L., Kwon, K., and Stivers, J. T. (2001) J. Biol. Chem. 276 42347-42354. 99. Asaeda, A., Ide, H., Asagoshi, K., Matsuyama, S., Tano, K., Murakami, A., Takamori, Y., and Kubo, K. (2000) Biochemistry 39 1959-1965. 100. Wyatt, M. D., and Samson, L. D. (2000) Carcinogenesis 21 901-8. 101. D'Ambrosio, S. M., Gibson-D'Ambrosio, R. E., Brady, T., Oberyszyn, A. S., and Robertson, F. M. (2001) Environ. Mol. Mutagen. 37 46-54. 102. Hill-Perkins, M., Jones, M. D., and Karran, P. (1986) Mutat. Res. 162 153-63. 103. Myrnes, B., Guddal, P. H., and Krokan, H. (1982) Nucleic Acids Res. 10 3693-701. 104. Sidorkina, O., Saparbaev, M., and Laval, J. (1997) Mutagenesis 12 23-8. 105. Kool, E. T. (2001) Annu. Rev. Biophys. Biomol. Struct. 30 1-22. 106. Kool, E. T., Morales, J. C., and Guckian, K. M. (2000) Angew Chem. Int. Ed. Engl. 39 990-1009. 107. Waters, T. R., and Swann, P. F. (1998) J. Biol. Chem. 273 20007-14.

PAGE 134

121 108. Porello, S. L., Leyes, A. E., and David, S. S. (1998) Biochemistry 37 14756-64. 109. O'Brien, P. J., and Ellenberger, T. (2003) Biochemistry 42 12418-29. 110. O'Brien, P. J., and Ellenberger, T. (2004) J. Biol. Chem. 279 9750-7. 111. Hill, J. W., Hazra, T. K., Izumi, T., and Mitra, S. (2001) Nucleic Acids Res. 29 430-8. 112. Yang, H., Clendenin, W. M., Wong, D., Demp le, B., Slupska, M. M., Chiang, J. H., and Miller, J. H. (2001) Nucleic Acids Res. 29 743-52. 113. Pope, M. A., Porello, S. L., and David, S. S. (2002) J. Biol. Chem. 277 22605-15. 114. Fasman, G. (ed) (1975) Handbook of Biochemistry. Nucleic Acids, Vol. 1, CRC Press 115. Selwyn, M. J. (1965) Biochim. Biophys. Acta 105 193-195

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122 BIOGRAPHICAL SKETCH Aarthy Vallur was born on 11.19.1975 in the gard en city of Bangalore, India. She obtained an undergraduate degree in nutrition and dietetics and a masters degree in biochemistry and molecular biology from the University of Madras Madras, India. She joined the interdisciplinary program in biomedi cal sciences in the University of Florida in 2000 for graduate studies. She was awarded an alumni fellowship to continue her research and obtain a PhD degree under the mentorship of Dr. Linda Bloom.


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Title: Kinetic Analysis of the Contribution of Base Flipping to the Substrate Specificity and Catalytic Activity of Human Alkyladenine DNA Glycosylase
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KINETIC ANALYSIS OF THE CONTRIBUTION OF BASE FLIPPING TO THE
SUBSTRATE SPECIFICITY AND CATALYTIC ACTIVITY OF HUMAN
ALKYLADENINE DNA GLYCOSYLASE















By

AARTHY C. VALLUR


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Aarthy C. Vallur
































This work is dedicated to my family whose unshakable belief in me made me aspire.















ACKNOWLEDGMENTS

My gratitude to my parents exceeds expression. They always instilled in me the

will and desire to achieve, aspire and hope in my abilities. I am indebted to my husband

for being an enormous source of support and encouragement through the trying years of

graduate school. I would also like to acknowledge the mentorship of Dr. Linda Bloom,

for the countless lessons I learnt from her and the very rewarding years I spent in her lab.

Beyond that, acknowledgements will not be complete without recalling the inspiration I

derived from my excellent uncle and aunts, all my teachers through school and college

and my dear friends, notably, Balaji Krishnaprasad of Gainesville.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .................................................... ............ .............. viii

LIST OF FIGURES ......... ............................... ........ ............ ix

ABSTRACT ........ ........................... .. ...... .......... .......... xii

CHAPTER

1 BACKGROUND AND SIGNIFICANCE...................................................................

The Problem of Chemical Reactivity of Bases..................................... ................. 1
Base Excision Repair--All Roads Lead to DNA ........................................ ...............3
Patch Size in B ase Excision R epair.................................... ........................... ......... 6

2 HUMAN ALKYLADENINE DNA GLYCOSYLASE- THE MASTER
F L IP P E R ............................... ......... ... ......................... ................. .. 9

AAG Knockout Mice and Implications for Repair................................ .................. 11
Diversity in Substrate Choice--Uniqueness of AAG.............................................. 14
Crystal Structure of AAG and Implications For Catalysis.............. ...................15
Relevance of Studying Flipping in AAG.................. ............................................ 20
Role of Tyr-162 in Flipping ............................................. .................. .... 20
Locating Substrates in DNA--Needle in a Haystack................ ..................22

3 EXPERIMENTAL PROCEDURES...................................... ......................... 24

Cloning and Expression of Human Alkyladenine DNA Glycosylase A79
(AAGA79) and Its Mutants....................... ............... ............... 24
Subcloning of hAAGA79-E125Q into pET-15b Vector....................................24
Purification of Human Alkyladenine DNA Glycosylase A79 and Mutants ...............26
Synthesis and Purification of Oligonucelotides..............................................28
Radio-Labeling of Substrates and Annealing to Complement.................................29
Single Turnover Excision Assay For Glycosylase Activity ....................................30
Multiple Turnover Assays for Glycosylase Activity ............ ........................32
Electrophoretic Mobility Shift Assay (EMSA) for AAG binding activity ...............32
Melting Temperature (Tm) Measurements for Duplex DNA...............................33









Fluorescence Assay for Ethenoadenine-AAG Binding and Excision ......................34
Stopped-Flow Fluorescence to Observe Flipping of sA by AAG..............................35

4 EFFECTS OF HYDROGEN BONDING WITHIN A DAMAGED BASE
PAIR ON THE ACTIVITY OF WILD-TYPE AND DNA-INTERCALATING
MUTANTS OF HUMAN ALKYLADENINE DNA GLYCOSYLASE ...................37

D N A Substrates and Sequences........................................... ........................... 39
Mutations to Tyr-162 and Projected Consequences............. ......... .. ......... .....40
Base Excision and DNA Binding Activities of the Y162S Mutant............................41
Activity of the Y 162F M utant ................ ......................................... ............... 43
Base Excision by the Y162F Mutant....... .......... ......... ........ ............... 43
DNA Binding Ability of Y162F M utant ........................ ......................48
Implications of Flipping in the Catalytic Efficiency of AAG................. .......... 51
A Two-Step Selection Model for AAG Activity .................... ......... ............... 54

5 ACTIVITY OF HUMAN ALKYLADENINE DNA GLYCOSYLASE IS
SENSITIVE TO THE LOCAL SEQUENCE CONTEXT OF THE DAMAGED
B A SE ........................... .................................... ...........................56

DNA Substrates indicating Base Stacking and Hydrogen Bonding Partners to
H x ....................................... .. .............. ..... ............................. . 5 7
Base Stacking and Hydrogen Bonding Effects on Hx.................... ................59
Effects of Hydrogen Bonding Partners on Hx Excision in the Strong and
W eak Base Stacking Context.......................... ........ .. ..........................60
Effects of Base Stacking Partners on Binding to Hx Substrates by AAG...........63
Effects of Hydrogen Bonding Partners on Binding to Hx in the Strong and
W eak Base Stacking Context......................................... ......................... 64
Base Stacking and Hydrogen Bonding Effects on sA ............................................66
Effects of Base Stacking and Hydrogen Bonding Partners on sA Excision
b y A A G .................................... .... ...... ........... .......... ............... 6 6
Effects of Base Stacking and Hydrogen Bonding Partners on Binding to
sA Sub states by A A G ...................................................................... ...............6 8
Melting Temperatures of Hx and EA Substrates................. ..................68
Sequence Context Effects and Implications for AAG Activity..............................70

6 ACTIVITY AND STABILITY OF AAG DURING ASSAYS ..............................75

Loss of AAG Activity Can Contribute to Reduced Catalysis ...................................76
Multiple Turnover of Hx Is Dependent on the Base Pairing Partner .........................81
Multiple and Single Turnover of sA Present Different Pictures ................................84
Optimization of Assay Conditions for Maximum AAG Activity .............................86
Conclusions about Activity and Stability of AAG During Assays............................89









7 FLUORESCENCE ASSAYS TO OBSERVE BINDING AND EXCISION OF
ETHENOADENINE BY AAG IN REAL TIME ............................. .................91

Fluorescence of sA During Binding by AAG ........................................ ........... 92
Blueshift of sA Emission upon Binding by E125Q......................... ............... 94
Fluorescence of sA upon Excision of sA by AAG................................ .........96
Stopped-Flow Analysis of sA Flipping by AAG .......................................................98
Contribution of Fluorescence Experiments to Understanding of AAG................. 100

8 DISCUSSION AND FUTURE DIRECTIONS..................................................102

The Night-Watchman Model for AAG Activity ...................................... 103
Tyr-162 and the Flipping Equilibrium .............. .. ................ 104
Stability of Hx and the Flipping Equilibrium .......... ......................................106
Not All Bases are Born Equal .............................. ............... 108
Future D directions ................ ............. .. .. .... .. ................. .... ... .... .... .......... .... 110
Completing the Need for a Real Time Assay to Measure Flipping ..................110
Continuing Research on Sequence Context Effects ............... ...............111
Possible Strategy to Explain sA Excision ...................................................... 111
Coordination of the Activity of AAG to Other Downstream Steps in BER ..... 113

LIST OF REFERENCES .......................................... .................. ... .......... 15

BIOGRAPHICAL SKETCH .............................................................. ...............122


SKETCH ............................................................. ............... 122
















LIST OF TABLES


Table page

4-1 Sequences of DNA substrates and positions of damaged base pairs .....................40

4-2 Observed excision rates and relative activities of AAG and Y162F mutant ..........47

5.1 Melting temperatures of Hx and EA substrates and corresponding single turnover
excision rates ...................................................................... .......... 70

6.1 Comparison of pre-incubation of 160nM AAG at 370C with product formed and
rates of product formation with 5nM THxA T .......................... ... ............... 78

6.2 Comparison of pre-incubation of 40nM AAG at 370C with the product formed
and rate of product formation with 5nM THxAT ............... ............... 81

6-3 Observed rates of multiple to single turnover titration assays of AAG with 5nM
EA T ............................................................................. 8 6

6.4 Comparison of the effects of pH and salt on AAG activity and stability with 5nM
G H x C T .......................................................................... 8 8
















LIST OF FIGURES


Figure p

1-1 Structures of damaged bases, commonly encountered in DNA ..............................2

1-2 Mechanism of action of mono-and bi-functional DNA glycosylases ....................5

1-3 Base excision and repair by different pathways............................................ ............ 7

2-1 Basal prom other region of the AAG gene ............................................................. 11

2-2 Bases found to be in vitro substrates for AAG ................ .............. ..................... 15

2-3 Residues that intercalate into the minor groove when AAG flips the pyrrolidine
ab asic an along ...................................................... ................. 17

2-4 Crystal structure of AAG bound to a pyrrolidine abasic nucleotide...................... 18

2-5 Crystal structure of E125Q bound to A........................................ ............... 20

3-1 The map showing the multiple cloning site and the other features of the pET-15b
vector. .............................................................................................25

3-2 A schematic of the excision assay and a sample gel for resolution of products
fro m su b states ..................................................... ................ 3 2

3-3 Fluorescent properties of 100nMsA when in double stranded DNA and after
excision by 400nM AAG at 370C for 60 minutes................ ............... 35

4-1 Chemical structures of hypoxanthine and 1, N6-ethenoadenine paired with
thym ine and difluorotoluene. .............................................................................. 39

4-2 Projected differences in intercalation ability of Ser-162 and Phe-162 when
compared to the wild type residue, Tyr-162, based on the crystal structure of
A A G bound to A .................. ...................................... .. ........ .... 41

4-3 Electrophoretic mobility shift assays to measure the affinity of the Y162S mutant
for DNA containing an sA*T or a Hx*T base pair. ...............................................42

4-4 Plots of time courses for Hx excision by wt AAGA79 and Y162F.......................45

4-5 Plots of time courses for sA excision by wt AAGA79 and Y162F.......................46









4-6 Control glycosylase assay to show that base pairing with F does not make A or G
excisable by AAG. ..................................... .... ... .... ... ... ............48

4-7 Binding of wt AAGA79 and the Y162F mutant to DNA containing Hx*T and
H x *F b ase p airs.................................................. ................ 5 0

4-8 Binding of AAG and the Y162F mutant to DNA containing sA*T and sA*F base
p a irs. .............................................................................. 5 1

5-1 Chemical structures of Hx and EA base paired to thymine, diflorotoluene and
cytosine..................................... ................................... ......... 58

5-2 Single turnover excision of Hx opposite T with T-A and G-C base stacking
partners. ..............................................................................60

5-3 Single turnover excision of Hx with T-A and G-C base stacking partners opposite
non-hydrogen bonding base pairing partner, F ................................ ............... 62

5-4 Single turnover excision of Hx with T-A and G-C base stacking partners opposite
W atson- Crick hydrogen bonding partner, C. ................................... ............... 63

5-5 Electrophoretic mobility shift assays to measure binding of AAG to DNA
containing Hx in different sequence contexts. .............................................. 65

5-6 Single turnover excision of sA with G-C stacking partners .......................... 67

5-7 Single turnover excision of sA opposite C with G-C base stacking partners.. ........67

5-8 Binding of E125Q to EA substrates with G-C stacking partners............................68

6-1 AAG death assay under single turnover conditions............. ................................. 77

6-2 AAG death assay under single turnover conditions..........................................80

6-3 Multiple turnover of Hx*T and Hx*F with G-C stacking partners .......................82

6-4 Multiple turnover of Hx*T and Hx*F with TG stacking partners ..........................83

6-5 Multiple to single turnover titration of AAG with A T. ........................................85

6-6 Effect of pH and salt on excision of Hx*T........................ ......... ...... ......... 88

6-7 Effect of BSA on AAG stability and activity...... ......... .................................. 89

7-1 The 500 second time-based fluorescence of sA when bound by increasing
concentrations ofE 125Q ............................................................ .............. 93

7-2 Emission spectra of 100 nMFA substrates, after adding 25 to 800 nM E125Q at
room tem perature. ........................ ......... .. .. ..... ............... 94









7-3 Emission scans of increasing concentrations of E125Q only when excited at
320 nm. .................... .. ........ .......... ......... 95

7-4 Emission spectra of 100 nM TEG with 100, 200 and 80 OnM Y162S. ...................96

7-5 Time- based increase in sA fluorescence upon excision by 800nM AAG at
370C. .............................. ................ ......... 98

7-6 Stopped-flow analysis of sA flipping by AAG. ...................................100

8-1 The Night-watchman model for AAG activity. ..................................... 104

8-2 Compromised flipping due to Tyr-162 mutation. ............. ............. ........... 105

8-3 Effect of Hx stability on the Night-watchman model.............................................108

8-4 Effect of EA excision on the Night-watchman model ...........................................109

8-5 A hypoxanthine* zebularine base pair. .............. ............. ............ ............111

8-6 Crystal structure of the AAG-EA complex showing the proximity of Met-169
to the etheno bridge of the flipped out EA in the active site................. ............... 113


e................................ 113















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

KINETIC ANALYSIS OF THE CONTRIBUTION OF BASE FLIPPING TO THE
SUBSTRATE SPECIFICITY AND CATALYTIC ACTIVITY OF HUMAN
ALKYLADENINE DNA GLYCOYSLASE


By

Aarthy C. Vallur

December 2004

Chair: Linda B. Bloom
Major Department: Biochemistry and Molecular Biology

Human alkyladenine DNA glycosylase (AAG) removes a variety of alkylated and

deaminated bases from double stranded DNA to initiate base excision repair of damaged

adenines. The crystal structure of AAG shows that the enzyme uses a characteristic base

flipping mechanism and does so by using Tyr-162 to intercalate through the minor

groove and occupy the space vacated by the flipped out substrate. The purpose of this

dissertation is to further understand the contribution of base flipping to the specificity and

efficiency of AAG.

A Y162S mutant showed undetectable activity on hypoxanthine (Hx) and 1, N6-

ethenoadenine (eA) substrates. A Y162F mutant showed 2-fold reduced activity on Hx

but the activity of the mutant was rescued by a "DNA mutation", in which the thymine

(T) opposite Hx was replaced by diflorotoluene (F) with which Hx cannot base pair. The










reduced activity of the Y162F mutant on sA was not rescued by the same "DNA

mutation".

Other changes were made to affect the stability of Hx in DNA in an attempt to

understand the importance of flipping to AAG activity on Hx. When HxoT was placed

between strong base stacking G-C partners, excision of Hx was reduced and required 4

times the AAG needed to saturate the excision of the same amount of HxoT placed

between weak base stacking T-A partners. When T was replaced by F, excision of Hx in

both sequence contexts was enhanced, but the enhancement with the G-C stacking

partners was greatest, up to 10-fold and enhancement with T-A stacking partner was

modest. When T was replaced by C with which Hx forms Watson- Crick bonds excision

was poor and the stacking partners did not matter.

The above results went on to show that AAG could use the Tyr-162 mediated

flipping mechanism to specifically bind and use the flipping mechanism as an important

step in recognizing the substrate. Factors that affect the flipping equilibrium which is the

first step in catalysis, like the stability of the damaged base in DNA will also affect the

capacity of the enzyme to remove the damaged base, ultimately. The sequence context of

the damaged base may be a determining factor in its recognition by DNA glycosylases

and hence in the initiation of its repair by the base excision repair pathway.














CHAPTER 1
BACKGROUND AND SIGNIFICANCE

The Problem of Chemical Reactivity of Bases

The bases in DNA can undergo chemical modifications which can alter the genetic

code and cause disturbances in the helical structure of DNA. Like any unsaturated,

heterocyclic compound, a base in DNA can fall prey to attacks by water, reactive oxygen

species and many endogenous and environmental agents (1-6). These chemical changes

account for around 20,000 damages/cell every day. Almost a quarter of these arise due to

the hydrolysis of the N-glycosidic bond between the base and the deoxyribose sugar

leading to abasic or apurinic/apyrimidinic (AP) residues in DNA. Purines are more

susceptible to hydrolysis of the N-glycosidic bond at an in vivo rate of >4500

depurinations/ cell (3, 7) whereas depyrimidination by hydrolysis occurs at a rate of less

than that of depurinations. Abasic sites are extremely mutagenic and cytotoxic since they

can lead to replication blocks and DNA strand breaks if not repaired (8). Deamination is

another consequence of hydrolysis in normal bases.

Oxidation is a major source of damage to bases. DNA bases have electrophilic

carbons that can undergo oxidation to form mutagenic lesions, primary of which is 7,8-

dihydro-8-oxoguanine (8-oxoG) (6, 9). Pyrimidine glycols and formamidopyrimidines

are also products of oxidative damage (Figure 1-1). Oxidative metabolism leads to the

accumulation of free radicals and products of lipid peroxidation like aldehydes which can

form etheno adducts with adenine, guanine and cytosine (3). Alkylation damage is also









brought about by S-adenosyl methionine, an abundant methyl donor in cells, which can

react with the ring nitrogens of purines (3) (Figure 1-1).

Deamlnatlon: Alkylatlon:





CH,
Uracil Hypoxanthine 3-melhyladenine


Rextew oxygen IpWldn:



N C14
N'H ^ 0 eOH
N O N T I, O 01-1

8-o,~auanine 4,6-dliamirIO 5 Thyminet glycol
formanopyrimidine



Figure 1-1. Structures of damaged bases, commonly encountered in DNA. Deamination,
alkylation and oxidation are chemical changes that contribute to damaged
bases in DNA (See text)

Other cellular agents that can react with bases include nitric oxide and its

derivatives. Nitric oxide (NO-) is an important second messenger in the body and is

involved in many functions including pathways signaling neurotransmission and arterial

wall relaxation (10). Nitric oxide is highly reactive with oxygen and forms nitrous

anhydride which can deaminate the primary amino groups of cytosine, 5-methyl cytosine,

adenine and guanine (11, 12). NO induced deamination of cytosine, 5-methyl cytosine

and guanine can lead to GC-AT transitions, which are the primary mutations seen with

NO-. of the three, guanine has the greatest susceptibility to NO- induced deamination,









which modifies dG to xanthine (13). NO- induced deamination of dA to hypoxanthine,

which can result in AT-*GC transitions. Spencer etal observed significant accumulation

of xanthine and hypoxanthine as the predominant lesions and a significant number of

single strand breaks in HBE-1 cells, a human bronchial cell line, when the cells were

exposed to ImM nitrite at times greater than 60 min (14). More in vivo studies with

cultured muscle nervous tissue cell lines, in which nitric oxide has important functions is

needed to learn about the degree and prevalence nitric oxide-induced deamination.

Base Excision Repair- All Roads Lead to DNA

The base damages discussed above are small chemical changes that do not disrupt

the structure of DNA drastically. But the problem of accumulating even small damages in

a vast sea of base pairs that make up the genome posts many challenges to the integrity of

the genome. Many pathways have evolved to repair damaged DNA and restore genomic

integrity, of which the repair of small, chemically damaged bases falls on the base

excision repair pathway (BER) (15-20). Initiation of BER is accomplished by a damage-

specific DNA glycosylases, which recognize its substrate and then excise the Cl'-N

glycosidic bond between the base and the sugar (Figure 1-2). This leaves behind an

abasic residue in DNA, which is then processed by an AP endonuclease to create a nick

5' to the abasic site. DNA glycosylases are thus very important in sustaining base

excision repair and ensuring its success. Up to eight human proteins have been reported

to have DNA glycosylase activity. The substrate specificity of glycosylases is wide, with

some glycosylases being selective for one substrate only while others have a broader

substrate range. Examples for glycosylases specific for one damaged base only are UDG

(uracil) and hOGGI (8-oxoguanine) while hAAG which can act on various adenine

lesions is a good example for glycosylases that can act on a group of substrates (16). In









addition to having Cl'-N glycosylase activity, some glycosylases also have an additional

AP lyase activity. These are called bifunctional glycosylases in contrast to the former,

which are monofunctional glycosylases.

Bifunctional glycosylases, also called the Class I AP endonucleases, utilize an

enzyme amino group as a nucleophile to form a Schiff- base intermediate with the abasic

site which then undergoes either a P or a 0-6 elimination reaction to leave a nick. 3-

elimination leaves an a-P unsaturated aldehyde with the 3'- phosphate and when followed

by 6-elimination (Figure 1-2), leaves just a 3'- phosphate residue which cannot be

extended by a polymerase (21). These 3'- termini can be acted on by a Class II AP

endonuclease, which processes the 3'- phosphate residue to leave a 3'- OH residue which

can then be extended by a polymerase. Repair is then completed when a DNA

polymerase replaces the nucleotide and a DNA ligase seals the 3'-OH and 5'- phosphate

termini. Some bifunctional glycosylases like the hOGG1 and the bacterial FPG also have

deoxyribophosphatase (dRPase) activity which can hydrolyze the abasic sugar- phosphate

residue (22). The major Class II endonucleases in E. coli are Exo III and Endo IV, Exo III

being responsible for 90% of the activity under normal physiological conditions (23, 24)

while Endo IV takes over during periods of oxidative and nitrosative stress (25, 26). The

mammalian homolog of Exo III is the human AP endonuclease I ( aka APEX, APEI, Ref

I), which is responsible for 95% of AP endonuclease activity in humans (22, 27, 28).

After the action of the Class II endonuclease in humans, deoxyribosephosphate

lyase (dRPlyase) activity and synthesis to fill the gap is carried out by polymerase 1 (Pol

0). After this, DNA ligase III, which interacts with Pol 1 through the XRCC 1 protein,

seals the nick to restore the original sequence in DNA. It is significant to note that Pol P









lacks the proofreading activity of replicative polymerases and is prone to a relatively high

frequency of errors during incorporation, to the tune of 1 mismatched nucleotide per

3000-5000 residues (29). These errors occur despite an induced fit mechanism in the

active site of Pol P. Two combined events ensure error free gap filling by Pol P. First,

DNA ligase III discriminates against joining ends with a 3' mismatch thereby, allowing

for excision of the wrong residue and replacement of the mismatched residue (30).

Second, just like replicative polymerases act as holoenzymes with separate subunits

encoding varied functions, a distinct human 3' exonuclease can cleave the mismatched 3'

residue and allow for replacement. Recently, a human homolog of the E. coli Pol III

holoenzyme component, Dna Q exonuclease, has been identified, which can correct Pol P

errors during BER and hence can be a major candidate for error free repair (31). Such a

proof- reading step will guarantee that correction of endogenous DNA damage does not

by itself contribute to a high frequency of mutations.

DN (E O NA0 B DN-D.



atAve H-0ai DNA gcys DNA l OH
1 0 $H













3 steps illustrate excision of the Cl-N glycosyi bond by nucleophilic attack by
activated water in mono- functional DNA glycosylases like UDG. Steps 4- 8


and Jiricny, J, Bioessays, 23:270-28], 2001)
andJiricny, J, Bioessays, 23:270-281, 2001)


f the Cl-N glycosyl bond by nucleophilic attack by
activated water in mono- functional DNA glycosylases like UDG. Steps 4- 8
illustrate the formation of a Schiff-base intermediate between a lysine residue
and the sugar followed by P-6 elimination.( Figure adapted from Scharer, O.D
andJiricny, J, Bioessays, 23:270-28], 2001)









Patch Size in Base Excision Repair

An alternative BER pathway to the single nucleotide replacement synthesis carried

out by Pol 0 has been observed in eukaryotes. This pathway involves the replacement of

around 4-19 nucleotides by DNA polymerase mediated strand displacement (32, 33) and

has been seen to require FEN1 and PCNA (34). One possible need for this long- patch

pathway could be the presence of termini which are resistant to the dRP lyase activity of

Pol 0 (35, 36). In this case, a more processive polymerase may cause strand displacement

and leave FEN1 to cleave the flap. The resulting nick is sealed by DNA ligase I. In

reconstitution assays it was shown that, in the presence of PCNA and FEN1, Pol 0 can

also carry out long- patch repair (32, 37, 38), suggesting that the role of PCNA may not

be to support processive replication, but activation of FEN 1 (37). There is speculation

that the BER initiating DNA glycosylase may influence the choice of patch size.

An added complexity is the observation that other factors may influence the patch

size. Sung and Mosbaugh used a closed, circular plasmid with a site- directed uracil or

ethenocytosine (sC) in E coli and studied the resulting patch size (39). In reconstitution

experiments with various BER proteins, they discovered that DNA ligase mediated end-

joining was the slowest step and also observed, short, long and very long patch repair.

Increasing the ligase to polymerase ratio biased synthesis towards short- patch repair.

With long time periods, longer patches (> 20 nucleotides) were observed (39).

The idea that the steps in BER are coordinated has been given a lot of thought due

to the complicated interplay of proteins and mounting evidence delineating the

interactions between enzymes catalyzing successive steps. Interactions between APE1

and Pol 0 (40), Pol 0 and XRCC 1 (41, 42), Pol 0 and ligase 1 (43) and XRCC1 and

PARP (44) have been reported. The role of PARP in BER may be accidental but









nevertheless important, as it is known to be a sensor for nicks in DNA and interacts with

XRCC1 (44, 45). Moreover, PARP knockouts are highly sensitive to methylnitrosourea,

which leaves behind BER substrates in DNA (46). There is also evidence for some

functional interactions between glycosylases and APE1. This step- wise coordination

may be important in bringing substrates and enzymes together and also protecting the cell

from the harmful abasic sites that accumulate as intermediates in BER.


Bifunctional
DNA glycosylase

S/En

AP Lyase



D
AP Endonuclease C

ULTTTTI; iiC
1 1 D
IIMMMMM^D


SMonofunctional DNA glycosylase


SAP endonuclease
)rrn rTTr r

DNA polls or PCNA



iNApolp 4 I FEN1W


& RF-C



& PCNA


Figure 1-3. Base excision and repair by different pathways. Base excision, initiated by a
damage specific DNA glycosylase can lead to resynthesis by either the short
patch pathway, in which one nucleotide is resynthesized or long patch
pathway, in which anywhere from 4-20 nucleotides are resynthesized. Mono-
and bi-functional glycosylases leave behind different 3' termini which are
processed by AP endonucleases or Pol P. Abbreviations: XRCC1- X-Ray
Cross Complementation protein, PCNA- Proliferating Cell Nuclear Antigen,
FEN1- Flap-Endonucleasel, RF-C- Replicating Factor- C.


UNA OIlB nTntrrrr

DNA ligase III/XRCC1


SHORT-PATCH PATHWAY


SDNA ligase I


LONG-PATCH PATHWAY









Though it is reasonable to assume that DNA glycosylases are localized to the

nucleus where they function, it is also important to address the problem of damage to

mitochondrial DNA, especially given its proximity to the oxidative phosphorylation

system. It has been found that many eukaryotic DNA glycosylase mRNAs like those

coding uracil DNA glycosylase (UDG) (47), human 8-oxoguanine DNA glycosylase

(hOGG1) (48) and the human MutY homolog (MYH) (49) are alternatively spliced to

encode nuclear and mitochondrial versions of the protein (50). Two additional DNA

glycosylase activities have been reported to be localized only to mitochondria, MtODE

and MtGendo (51, 52). These act on oxidatively damaged bases. More research is needed

to reconstitute the BER pathway in the mitochondria and identify other proteins and

splice variants for other components of the pathway.














CHAPTER 2
HUMAN ALKYLADENINE DNA GLYCOSYLASE- THE MASTER FLIPPER

Alkylating agents constitute one of the major offenders of the integrity of bases in

DNA. Alkylated purines are mutagenic, especially susceptible functional groups in

purines being the N7 of guanine and the N3 of adenine. Though commonly resulting

from environmental and chemotherapeutic agents, alkylation damage can also result as a

by- product of cellular activities. A unique suicidal enzyme, 06-methylguanine methyl

transferase directly reverses O6-methyl guanine lesions which are highly prone to

mispairing with thymine (53, 54). Most other alkylated bases are removed by specific

DNA glycosylases and alkylated base- specific glycosylases have been cloned in almost

all species studied, including bacteria, yeast, mice and humans. With the discovery of

UDG by Lindahl and coworkers in 1974 (55), came the awareness about the need for

more glycosylases to play a role in base excision repair. Research since has identified E.

coli glycosylases responsible for removing 3-methyladenine (3-meA) and other cytotoxic

alkyl purines (56-58). In 1982, Goldthwait and co- workers identified two distinct

glycosylases responsible for excising 3-meA, a 3-meA DNA glycosylase I constitutively

expressed from the tag gene and a 3-meA glycosylase II which is encoded by the

inducible alkA gene (59). The tag gene product was specific for 3-meA while the alkA

product showed a broad substrate range including 3-meA, 3-meG and 7-meG.

Subsequently, alkylpurine glycosylase activities have been identified and purified from S.

cerevisiae (MAG), mice (MPG) and humans (AAG). Alkyladenine DNA glycosylase

(AAG) was identified as the human equivalent ofE. coli AlkA and could complement tag









and alkA deficient strains. It was independently characterized by three groups who

alternatively named it as alkyladenine DNA glycosylase (AAG) (60), methylpurine DNA

glycosylase (MPG) (61) and alkyl-N-purine DNA glycosylase (ANPG) (62). It has been

the only alkyl purine glycosylase discovered in humans so far. In 1993, Vickers et al

localized the human AAG gene between the a- globin gene cluster and the telomere of

the short arm of chromosome 16 (chrl6p) (63). Working with a colon adenocarcinoma

cell line (HT29) and a human erytholeukemia cell line (K562), they identified the gene as

comprising five exons whose representation differed in the 5' end from the cloned cDNA

reported earlier by Samson et al (60). Rafferty and co- workers, in 1994, screened a Xgtl 1

human placental cDNA library with a probe derived from the original sequence and

identified two alternative cDNAs, which differed in seven N-terminal residues (64). They

concluded that these were likely to be splice variants differing in exon usage and could be

important in accounting for some properties such as cellular localization and binding

properties of the protein. In a search for more information on the expression of AAG, O

Connor's group located the basal promoter within 80 bases of the start codon of exon 1 in

HT-29, K562 and 3T3 cell lines (65). They also identified some putative transcription

factor binding sites in the promoter region, including those for N-MYC, SP1, USF-1 and

CBP. In super shift assays, N-MYC and SP1 were found to bind and super shift the

promoter region (Figure 2-1). It was also reported that expression of the AAG gene was

cell- cycle dependent. The expression was seen to increase during G1, remain elevated

during synthesis and then decrease to basal levels. This was consistent with DNA

synthesis and the need for repairing replicating DNA. Other BER enzymes like APE1 and







11


Pol 0 have been found to be induced upon accumulation of DNA damage. Whether AAG


is also induced requires more extensive in vivo studies to ascertain.

Sac 1
-700 AACCCAGAAC G~OGGOAGC TCCC TC. e ATCcGr GoC GGAAAGAC AG NTOOMTC TTCGCAGGP GCAGAATOC G CO2CGCTCC TCCTT'POIC -01L
TTGCGTCWIG CMCCIGTC7G ACOGAfICC TMAGMGACCO CCTITCTCCC TACCCCCOAG ACGATirCTC CGIrTAGGC GCGGCGAGGsG AGGAJAAGGA

-600 CCCCGtCCA CCT-G Z AC CCTCCT CCA GOaGGCGr03G CICCACA GOCTCTlXCTC CACTTCCTTO ATAAGGhACC OCWCCLCAC TCCGACCAG -501
GGGGCTYGrC GGCAXCIT OGGGCCCGTC CCcO:CCCO3 (QAGETOTT f?:'-r G=&AGGAOC TATCC. AG QCM3GC0GT ACOXCXGTC

-500 GMcOGMG GCCTCCTCMCA CGCTCACTCC GCTGGeaCC CACCG~AAAA CACTYJOGCG CCCGCCCCO TCCN AAAG CACGCCACT CCOCGG C -401
CCGAOCCCCG CGAGMGeTCC GCGaARnGG CGChGf CGTGCcrTTTT 0ThCAGACOC CGCaGGO AGG:S'IIC GorGONGGTGA GCWGCCG

-400 CCCTCGAGS TCCACTCAGO T'TCACGCG OCCCO;TtCTC TO3GTGTCT GACIrCCGGC TCCGGOTGGA OCTCTIG CCGOCAGGCG ACCAMGGCGC -30I
GGcGCACTCC AG3TGAGC hGKTG AACTCC CGOCeAfAGO AGWCCAGA crMGiaCGc A~CCACTAP CCrA(a;_R c GC-TCCGC TIGGTE-GCG

....... CCAAT o.....
-lo 300GG03C GcWXOlX ABCT'C ABGGG CGCCGOGCA CAGTCGGO CrCCOrAC CCCflGGToC CCCTTICCO CTITCGCCTC GCPOeCACCG -201
TCCGcocXC CCGrcArMGT rccrc 'ceGcCcor Ci;ACGCCCC GA4cMGWs GGG3TCCMAC G03AOJGC okGAAGCGAG CGrfOMrC C
**t,.q.

Rsr i ,, cattg USF-1
1 N-MYC -'- .____ CCAAT
-200 CATCGGCCC OGsAATCGOT OCOGACCX-G CGTG G CGC- TGiGAAAG ATCCACC~CT C ACCCCCcCOG Cc--crcCG r-. ... c ..* C,::. -101
GTAGCOCGG CCCTPT CCA QOCrCT=kC GCCACCCGC ACCTCC TAGOOGTPAGG TICACCOGGC GGC GG:GQiC ::-:i. .. --r .*
Avrl) / 9tg SP1 E2F
I I
-100 COC'TCAMTC CCCCCTTCTC CCOGCfTTC:GT CCCCTTCTG CGCAGI=GG GCTCCGCCCC GRTCCTACGGf GOTOTTCCGT GGTCUGGC TGCTGGGTC1 -1
GCCAGTGACG GG GAAIGM GGCCG AAOCA c O :AAiC GCGTCCCGS CG~GGCGG3a C~AO3ACCC CACCA3CAA CCAGCO=CCG ACGECCOGAG

+1 COCFCCCIGGTCOeAGrCCAC GCCCGCCGACCGCCOB atg ccc ;og cgc age ggg gcc cag
Met tro Ala Arg Ser Gly Ala Gin



Figure 2-1. Basal promoter region of the AAG gene. Putative binding sites for several
transcription factors like USF-1, SP1, N-MYC and CBP were noticed
(Adapted from Bouziane et al, Mutation Research, 2000, 461:15-29)

AAG knockout Mice and Implications for Repair

Questions remained as to the precise biological effects of methyladenines in the cell


and the possible role of protection by AAG activity. Given the broad substrate range


observed for the mouse 3-MeA glycosylase (MPG), homozygous AAG knockout mice


were generated by two groups and examined for phenotypes that can make the role of


AAG clearer. Engelward et al, reported in 1997 that AAG was the major 3-MeA


glycosylase in at least 4 different tissues, namely, liver, kidney, testes and lungs in these


mice (66). Mouse embryonic fibroblasts that were deficient in AAG were more sensitive









to Me-Lex, an alkylating agent that generates 3-MeA specifically in DNA, than the wild

type AAG producing fibroblasts. AAG was also found to be the only glycosylase

responsible for removal of Hx when tested in the testes, kidney and lung tissues. It was

also the major activity responsible for excising sA lesions, but it is interesting to note that

though no other sA glycosylase activity was noticed in testes and kidney, there was a

minor degree of sA excision noticeable in the lungs of the AAG-~- mice.

Elder et al also published the generation and effects of the AAG-- genotype (67).

They reported an increased frequency of MMS- induced mutations in the hprt gene of

splenic T- lymphocytes after a single MMS dose of 150mg/kg. These mutations were

mostly AT--TA transversions (47%) with some GC-TA transversions (27%). The

mutation frequency was about 10-fold higher than the background. When analyzed, they

found an accumulation of 3-MeA and methylated guanines in the hprt gene up to a 24hr

period after MMS treatment, but found that 06-MeG and 04-MeT which are also formed

at lower frequencies by MMS were efficiently cleared in the knock out (ko) cells lacking

MPG activity. A reason for the cytotoxicity of 3-MeA and 7-MeG are their labile

glycosylic bonds depurinating at a higher rate leading to abasic sites in the DNA. 06-

MeG and 04- MeT are highly prone to mispairing and creating single base changes. 06-

MeG and 04- MeT seem to be cleared by another redundant activity, possibly by the

nucleotide excision repair (NER) machinery or another DNA glycosylase. Elder et al

concluded that the importance of AAG may be more than the need to protect against 3-

MeA and 7- MeG. It may be more important to protect the genome from its other

substrates, Hx, which is highly mutagenic and can cause AT--GC transitions and eA,









which is also promutagenic. Both these lesions have stable glycosidic bonds and may be

the primary target for AAG.

One remarkable feature of both these knockout mice was that both strains were

active, viable, and fertile and did not show any other abnormalities, prompting the

conclusion that either the spontaneous lesions that are the targets of AAG were not lethal

when unrepaired or that other glycosylases/repair pathways can assist to handle these

lesions. This is important because there is evidence that, in yeast, NER can process some

AAG substrates and can also act on some other methylated substrates like 06-MeG. It

was recently reported that human AAG can interact with the human RAD23 proteins,

which are involved in recognition of damaged bases in DNA in NER and that the

interaction can functionally affect AAG binding and excision activity on Hx containing

DNA (68). Nevertheless, the AAG knockout model is the first glycosylase/BER

homozygous knockout model available to better understand the complex pathway.

Knockouts of other downstream enzymes in BER have proved embryonic lethal, possibly

due to the general requirement of enzymes like Pol 0 and APE1 for processing all

products of glycosylase action. These enzymes may also have other roles to perform and

any possible complementation in activity by the NER pathway may not be sufficient to

make up for the loss of these enzymes in the knockout cells. Loss of individual DNA

glycosylases, on the other hand, may be much easier to complement by other DNA

glycosylases or pathways. Hence, homozygous knockout models of individual DNA

glycosylases, like the AAG knockout mice discussed above, may be useful in generating

heterozygous crosses to study the effect of the BER machinery and its interplay with

other repair machineries.









Diversity in Substrate Choice- Uniqueness of AAG

E. coli AlkA, mouse and human AAG have all been found capable of removing a

diverse range of substrates unlike many other DNA glycosylases which are specific for

one damaged base only. AAG was first identified as a 3-MeA glycosylase and hence

named so (60, 62). But later, it was discovered to be able to remove 7-MeG, 8-oxoG, Hx

and eA bases in vitro (69-71). The AAG-- mice demonstrated that it was the major

glycosylase responsible for the removal of 3-MeA, Hx and eA. 8-oxoG may not be an

AAG target since it was removed efficiently in the AAG ko cells probably by its own

dedicated glycosylase, OGG1 (72). So being able to excise out at least 4 structurally

diverse substrates in the cell raises the question of whether catalytic efficiency is

compromised for substrate diversity. Methylated adenines and guanines can arise

spontaneously in vivo from the action of methyl donors like SAM. Methyl adenines are

unstable and spontaneously depurinate at a higher rate than normal purines, leading to

cytotoxic abasic sites in DNA, in addition to having the ability to block replication forks.

Hx is deaminated adenine, formed by the action of nitric oxides and its derivatives in the

cell. Nitric oxide is an important second messenger and is also released by activated

macrophages. Hx is highly mutagenic. eA on the other hand is formed by the action of

lipid peroxidation products in the cell and the action of the common hepatocarcinogens,

vinyl chloride and ethyl carbamate (62, 73, 74). In COS7 cells, 70% of EA lesions led to

mutations. So, the biological cost of compromised catalytic efficiency for substrate

diversity can be immense, making AAG a uniquely gifted enzyme. How one active site

can be tailor- made to fit these diverse substrates is difficult to imagine. Extensive

biochemical work done by our group show that, AAG-mediated excision is sensitive to










the base pairing partner of the damaged base, and the extent sensitivity varied with the

identity of the damaged base (75, 76).

NH2 0
H3C 0


ND N N N2 N NH2
H N N NH2 +
CH3 CH3

3-methyladenine 7-methylguanine 3-methylguanine

o o

N N
N NH -I NH

N N
N NH N NH2 H N NH2

hypoxanthine 8-oxoguanine 8-hydroxyguanine

0


N N NH N

HN N N
HN HH

1, N6-ethenoadenine ethenoguanines



Figure 2-2. Bases found to be in vitro substrates for AAG. 3-methyladenine, 7-methyl
guanine, hypoxanthine and 1, N6-ethenoadenine were found to be excised by
AAG in AAG-- cells and are italicized for emphasis.



Crystal Structure of AAG and Implications for Catalysis

The diverse substrate specificity of AAG and the fact that it is the only known

alkylpurine excising glycosylase makes it an interesting protein whose structure can not

only tell us more about general glycosylase action but also about the complicated nature

of substrate recognition used by AAG. Lau et al reported two crystal structures of AAG,









one bound to a pyrrolidine abasic site analog (77) and another of the catalytically inactive

AAG mutant bound to one of its substrates, ethenoadenine (78). Both have contributed

enormously to the understanding of AAG and have provided a tremendous boost to

research on AAG and its mechanism of action. The first reported structure was a 2.7A

crystal structure of an enzymatically active fragment of AAG lacking the first 79 amino

acids in the N-terminal, bound to a pyrrolidine abasic nucleotide, which is a potent

inhibitor of the glycosylase activity. This structure showed AAG as a single domain

containing seven a helices and eight 0 strands. 0334, in the core of the protein, protrude

as a p hairpin that inserts into the minor groove of the bound DNA, and displaces the

target nucleotide, in this case the pyrrolidine. The displacement is made possible by

insertion of Tyr-162, so that it intercalates in the space occupied by the nucleotide, which

is then flipped out of the helix into the enzyme's active site binding pocket. This flipping

mechanism was first identified in the cytosine methyl transferases (79, 80) and has also

been consistently observed as the binding mechanism of choice for BER proteins like

glycosylases (81-83) and endonucleases (84, 85).

In the AAG-DNA complex the B-form DNA duplex is kinked away from the

protein where Tyr-162 intercalates, by about 220 and is held by two clusters of basic

residues that contact the DNA backbone on either side of the flipped out residue. The

extent of the buried DNA surface, as measured with a 1.4A probe was 1034A. This is

similar to that observed with the UDG-DNA complex (81). The flipped out pyrrolidine is

looped out of the helix by rotation of the P-05' bond and the 03'-P bond of the

phosphodiester backbone on either side of the nucleotide. The intercalating Tyrl62

pushes out the opposite T19 residue 1.5 A into the major groove. No specific contacts









between the opposing T19 and enzyme residues are visible in the flipped- out nucleotide-

AAG structure. Met-164 and Tyr-165 located in the 0 hairpin, fill the minor groove and

push against the deoxyribose moieties of T19 and T9, thereby widening the minor groove

by almost 2 A, 3' to the flipped out pyrrolidine. This 3' distortion may help AAG to scan

the DNA unidirectionally.


Figure 2-3. Residues that intercalate into the minor groove when AAG flips the
pyrrolidine abasic analog (Pyrr 7). Tyr-162 is fully intercalated between
Guanine 6 and Thymine 8, while Thymine 19 (T19) is pushed into the major
groove. Met-164 and Tyr-165 widen the minor groove.






























Figure2-4. Crystal structure of AAG bound to a pyrrolidine abasic nucleotide. The
enzyme binds by intercalating Tyr-162 into the minor groove and flips out the
abasic nucleotide into the active site. Enzyme residues widen the minor
groove (see text) (Adapted from Lau etal, Cell 95, 249- 258, 1998)

The active site shows a central water molecule well positioned for a nucleophilic

attack on the glycosyl bond of the flipped- out nucleotide. It is linked by a hydrogen

bonding network with the pyrrolidine nucleotide and the side chains of Glu-125 and Arg-

182 and the main chain carbonyl of Val-262. Glu-125 acts as a general base by

deprotonating the water and thereby activating it for attack. Tyr-127 stabilizes Glu-125

by forming hydrogen bonds and may also stack against the base. The 2.1 A crystal

structure of AAG bound to eA gives a clearer picture of how a substrate is bound in the

active site. In this structure, Tyr-127 stacks against eA while another active site tyrosine,

Tyr-159 makes an edge to face stacking interaction with the eA. The flipped- out base

itself is rotated 850 about its glycosyl bond away from the double helix. It is remarkable

that these active site residues located in the buried core of AAG are conserved among the









other putative alkylation repair glycosylases identified in Borrelia burgdorferi (86),

Bacillus subtilis (87), Arabidopsis thaliana (88), and Mycobacterium tuberculosis (89).

The other important family of DNA glycosylases, of which E. coli AlkA, S. cereviseae

MAG and human OGG1 are members, binds DNA and flips out the target base through a

structurally conserved helix- hairpin- helix motif and use an aspartate as the catalytic

residue, though there is no obvious sequence homology in this family of glycosylases.

Whereas no AAG homologs have been noticed yet, in terms of its structural or sequence

details, the conserved active site residues point out that proteins with similar folds may

employ similar catalytic mechanisms, especially given the divergence of the glycosylases

that share these conserved residues, in contrast to the structural homology seen in the

HhH family of glycosylases.

The crystal structure also gives a hint of how AAG could achieve substrate

specificity given its diverse substrate range; yet exclude normal purines from its active

site. Positively charged substrates like 3-MeA and 7-MeG are electron deficient and

hence, they may be bound and excised by using the aromatic 7t-electron stacking strategy

in which, aromatic residues in the active site like Tyr-127 stack favorably with the

flipped out base, thereby stabilizing it for excision. This method of sandwiching electron

deficient bases between aromatic residues is a hallmark of many methylated base binding

proteins such as AlkA and several mRNA binding proteins (82, 90). Add to it the

intrinsic instability of the glycosylic bond in these bases, effective excision is more

probable. But this same strategy may not work for neutral bases like Hx or sA. The sA-

AAG crystal structure shows that EA is accommodated in the active site by a combination

of aromatic stacking and hydrogen bonds between the main chain amide of His-136 and









the N6 of the flipped out base. The stacking offers a lone pair of electrons which would

not be possible if the base was adenine. In the same manner, the 06 of Hx can accept a

hydrogen bond from His-136, although guanine can do the same, its N2 amino group will

clash with the side chain of Asn-169, thereby restricting access to both normal purines

but allowing both neutral substrates. This strategy for fitting the flipped nucleotide in the

active site allows AAG to both screen for the right substrate and accommodate only the

right substrate for excision, once flipped.


tJArq 182

4 Glu'Gtnl 25
35

Asn 169

STyr'A Tyr127








Figure 2-5. Crystal structure ofE125Q bound to EA (left panel). The black EA is flipped
into the active site, where it stacks between Tyr-159, His-136.and Tyr-127.
The active site is clearly shown in the right panel. The orientation of the
flipped out EA relative to the catalytic residue and the water molecule is
shown in a superposition of the active sites ofE125Q/ EA (green), wild- type
AAG/ EA (pink.) and wild- type AAG/ pyrrolidine abasic site (blue) (Adapted
from Lau et al, Proc. Nat. Acad. Sci. USA 97-13573, 2000)

Relevance of Studying Flipping in AAG

Role of Tyr-162 in Flipping

The flipping mechanism facilitated by insertion of Tyr-162 into the minor groove,

is indicative of both how tight binding is achieved by AAG and how specific binding









may be achieved. The limited DNA contacting surface seen in the crystal structure and

the absence of any specific contacts between the opposite base and enzyme residues also

increases speculation on how important Tyr-162 mediated intercalation is to the overall

efficiency of substrate binding by AAG. In contrast, E. coli UDG, which flips out uracil

from DNA, makes extensive contacts with the phosphodiester backbone of the DNA and

involves a "pinch-push-pull" mechanism to extrude the uracil from the DNA into the

active site (85). In this model, UDG, scans along the DNA using its leucine finger .When

a U-G wobble base pair is detected by the leucine, the Ser- Pro loops compress the

phosphodiester backbone extensively, kinking the DNA to almost 450, thereby pinching

the DNA to gain access to the uracil. This pinching gives the needed force for the leucine

to intercalate into the minor groove thus, "pushing" the uracil out of the helix where it is

"pulled" into the active site by specific hydrogen bonding interactions. The last two steps

can happen in either order. It is clear how protein- DNA interactions other than leucine

insertion can contribute to flipping uracil by UDG. But these interactions seem to be

largely missing from the AAG crystal structure, in which we do not see extensive

compression or kinking of the DNA to the extent seen with UDG. In addition, leucine

mutations to alanine or glycine reduced UDG activity to 10% and 1% respectively (91-

93). In a similar attempt to test the functional significance of Tyr-162 mutants and wild-

type AAG were expressed in a S. cerevisiae strain lacking the endogenous yeast MAG1

and tested for resistance against the alkylating agent MMS. In contrast to the wt AAG,

the Y162A mutant made yeast very sensitive to MMS, pointing out that maybe the

mutation may have rendered AAG unable to bind and hence excise alkylated bases. In

contrast mutations to the two other residues, Met-164 and Tyr-165, which are also part of









the P-hairpin, and which are shown to assist flipping by destabilizing the base pair next to

the flipped out base, did not confer MMS sensitivity to the yeast cells (78). Mutation of

Tyr-162 to Ser reduced AAG activity to undetectable levels, in kinetic studies done by us

(75). So the importance of Tyr-162 to the process of flipping could be significant and in

the absence of other factors that could affect DNA binding, must also contribute to the

overall catalytic efficiency of the enzyme in ways more extensive than what is known

from UDG.

Locating Substrates In DNA- Needle in a Haystack

The crystal structure offers no plausible explanation of how the daunting task of

identifying its substrates amidst millions of bases is accomplished in the vast genome

with its complex hierarchy of structures. It does make sense though, to expect AAG to

use Tyr-162 intercalation to scan the DNA or possible substrates. Since AAG substrates

are not known to distort the backbone extensively, a partial unstacking of the nucleotides

may be needed for AAG to identify its substrate, as proposed for AlkA (77). Once a

nucleotide is thus flipped, the disproportionate rigidity produced by Met- 164 and Tyr-

165 may help the protein progressively flip nucleotides in the 3' direction, thereby

scanning the minor groove for substrates. Once a base is recognized, the interactions in

the active site may include or exclude the base as a substrate for excision. This two- step

recognition may also help AAG preserve its substrate diversity. An added complexity

comes from the reports that, though AAG does not seem to make any specific contacts

with the opposite base, it is sensitive to the opposite base in terms of binding and excision

proficiency (75, 76). These findings further enhance the need to understand the

mechanism of AAG may achieve substrate diversity, given the different properties of the

substrates it excises.









A systematic analysis of the biochemistry of flipping and its effects will be very

useful in understanding the mechanism of action of AAG and inching closer to the

unsolvable riddle of how glycosylases find their substrates in the vast genome. This

dissertation attempts to address the issue of the contribution of flipping to the activity of

AAG. A systematic, biochemical approach to analyzing the effects of mutating Tyr-162

on flipping efficiency and the contribution of local DNA sequence context to flipping

efficiency was undertaken. The kinetic interpretation of the resulting observations reveals

important biological implications about the contribution of flipping to the specificity and

the catalytic efficiency of AAG (75). The conclusions drawn from this dissertation and

the reagents developed to conduct the research will be valuable in dictating further

studies on AAG mechanism and action.














CHAPTER 3
EXPERIMENTAL PROCEDURES

Cloning and Expression of Human Alkyladenine DNA Glycosylase A79 (AAGA79)
and Its Mutants

A deletion mutant of AAG that is missing the first 79 amino acids from the N-

terminus (AAGA79) was constructed using PCR, as already published (75, 76). Deletion

of this unconserved N-terminal region has been shown to have no effect on either base

excision or DNA binding activities of the enzyme, but the truncated protein is more

soluble at low ionic strength. All site-directed mutants were made in the coding sequence

of this truncated gene using the Transformer Site-directed mutagenesis kit (BD

Biosciences Clontech, Palo Alto, CA). The primers used to generate the desired

mutations were also engineered to contain a silent mutation that created a restriction site

to facilitate screening of clones.

Subcloning of hAAGA79-E125Q into pET-15b Vector

The hAAGA79 constructs were cloned into pET-14b vectors and were used to

transform E. coli BL21 (DE3) cells. The hAAGA79 -E125Q mutant was subcloned into a

pET-15b vector due to its incompatibility with the pET-14b vector. The pET-15b vector

has a multiple cloning site with a T7 promoter sequence and the lac operator sequence,

which can allow for more controlled expression of the cloned gene and hence help get

over the incompatibility problems seen with pET-14b. The E125Q gene sequence along

with an upstream ribosome binding site was subcloned into the Xbal and the BamH1

restriction sites (Figure 3-1). The vector was transfected into DH5-a cells and the plasmid











was isolated from the cells. The sequence and alignment of the E125Q gene was checked


by sequencing. All other AAG mutants were cloned into pET-14b vectors.


Multiple cloning
Bpu1102 1(267)
EcoR 115706 / BamH 1(319) Se
Aat 11(5635) Cla 1(24) Xho 1(324)
Ssp 1(5517) Hind 111(29 Nde 1(331)
Nco 1489
Sea 1(5193), Xba 1(428)
Pvu 1(5083) Cg I, J.J,
Pst 1(4958) Sph 1(691
EcoN 1(751)
Bsa 1(4774)
Ahd 1(4713)

MIu 1(1216)


AiN 1(423) pET-15b Apa 1(1427
(5708bp) AII ..


Hpa 1117221
BspLU11 ,;,.
Sap 1(3704)
Bst1107 1(3591) PshA 1(2061)
Acc 1(35 0)
BsaA 113572) Eag 1(2284)
Tth111 lo3565) N\ 1(2319)
120) BspM 1(2399)
BpulO 1(2926) BsM 1(2704)
MSC 1(2791)



T 7 proinoe primer 69348-3
Bl 7TT promoter lac operator X | risa
AGATCTCGATCCCGC AAATTAATACOAC TCACTATAGNOAATTGTA GCOUATAACAATTCCCCTCTABAAATAATTTTTO TTTAAC-TTTAAAAW lAI
ail 3HIsTaOg ir I .r TATACCATOGGCASCCCCATCATCATCATCATCACACAUgCZCCTGSTQCCGCGCGQGASCCATATOCTC AGAATCCSOCTSCTAACAAAGCCCOA
netG lySgr r ml aH I *I I lh IN I SIHi 5r*BrBIr yLyIuVa IPror I IrHfliS tLGEIU A u A I A* I m Lyli aArg
1Brl 1''2 I Ihrontblin T7 terminator
AAGGAAB!CTGAGTTGGCTBCTGCCACCGCT AGCAATAACTASCATAACCCCTTGGGCCTCTAAACBGGTCTTGAGQBGTTTTTTE
Iyg l II alLamuAlA IaAlalhrA~ IaluG iEnd
T7 lenlnalor primer #69337-3


Figur 3-1. The map showing the multiple cloning site and the other features of the pET-
15b vector. The multiple cloning site and its components are shown in the
sequence below the map. The restriction sites used to subclone the E125Q
gene, the Xbal and the BamH1 sites are highlighted in blue with arrows.


The transformed cells were plated on 2xYT plates containing 100tg/mL ampicillin


and grown for 16-20 hours at 370C. A 10-mL aliquot of fresh 2xYTcontaining 100jtg/mL


ampicillin was inoculated with a single colony of transformed cells and grown at 370C


with vigorous shaking (200 rpm) to A600 of 0.5-0.8 and then stored at 40C overnight. The









next day, the cells were spun down and washed two times with 2xYT- 100tlg/mL

ampicillin (10mL per wash) and then resuspended in fresh media. The washed cells were

used to inoculate 3 liters of fresh, autoclaved 2xYT- tlg/mL ampicillin. Cells were grown

with shaking (200rpm) at 370C to A600 =0.5. The cells were then cooled to 20- 250C in

the cold room. The cells were then induced with a 0.4mM final concentration of

isopropyl P-thiogalactopyranoside (IPTG) and allowed to express at room temperature

(20-250C) with shaking for an additional 8 hours. Cells were then harvested by

centrifugation at 5000rpm for 30 minutes at 40C using a JLA-200 rotor (Beckman). The

cell pellets were drained thoroughly and were stored at -800C until needed. The pellets

can be stored at -800C from at least 16 hours up to a week for maximum protein yield.

Purification of Human Alkyladenine DNA Glycosylase A79 and Mutants

The frozen pellets were weighed and resuspended on ice in cold buffer A (50mM

sodium phosphate, pH 7.4, ImM EDTA, 70mM KC1, 10% glycerol, 0.5% 2-

mercaptoethanol, 1 ltg/mL pepstatin A, 1 ltg/mL leupeptin, 0.1 [tg/mL PMSF)

Approximately 3mL of buffer per gram of pellet was used. The resuspended pellets were

then pooled into pre-chilled conical tubes and were lysed in a Aminco-SLM Instruments

French press after loading into a 40K cell (30mL capacity) and pressing at medium

(700psig) followed by maximum pressure (2550psig). The press was repeated again to

ensure thorough lysing. The pressed cells were immediately placed on ice and

centrifuged at 10, 000 rpm for 45 minutes in pre-chilled plastic tubes at 40C using a JA-

20 rotor (Beckman). The supernatant, containing the soluble protein was then pooled into

a pre-chilled conical tube and placed on ice. All further purification steps were carried

out at 40C and the protein fractions were always kept on ice.









The pooled supernatant was loaded on to a 200mL diethylaminoethyl (DEAE)

cellulose (Sigma) anion- exchange column using a peristaltic pump. The DEAE cellulose

was batch equilibrated with several changes of 125mM sodium phosphate( pH 7.4)

before packing until the material reached a pH of 7.4- 8.0. It was then packed in a column

and further equilibrated to pack and flow at a rate of 2mL/ min. using two column

volumes of buffer A without the protease inhibitors. After loading the supernatant on to

the column, the column was washed with one column volume of buffer A with protease

inhibitors. AAG which has a calculated pi of 9.1 will not bind to the DEAE and hence

elutes in the wash. This step is useful in removing the vast amounts of DNA from the

supernatant. The flow-through fractions were collected in pre-chilled conical tubes placed

on ice. They were then loaded at 2mL/ min onto a 5mL Hi-Trap sulfopropyl sepharose

(Hi Trap S) cation exchange column (Amersham Pharmacia Biotech) using an Amersham

Pharmacia Biotech FPLC system. The column was previously washed in 2 column

volumes of 1M KC1 to strip any protein stuck and then equilibrated in 5 column volumes

of buffer A. AAG binds to the column under these conditions. An 80mL gradient from 70

to 500mM KC1 in buffer A was then used to elute AAG from the Hi trap column. AAG

usually eluted at approximately 250-300mM KC1. Fractions of 1.8mL were collected and

stored on ice. The fractions eluting at 250- 300mM KC1 were analyzed by SDS-PAGE

for the presence of AAG and those fractions that contain AAG were pooled and

concentrated to 1- 2mL in an Amicon Ultracentrifugation Stirred cell (Model 8010)

using a 5000D cut-off Polyethersulfone membrare, wetted in nanopure water. The

concentrated fractions were then loaded on to a 125mL Sephacryl S-200 HR gel filtration

column, at 0.5mL/ min using an Amersham Pharmacia Biotech Gradifrac system.









Previously, the column was isocratically developed at ImL/ min with buffer B (25mM

Tris, pH 7.5, 200mM KC1, ImM EDTA, ImM dithiothreitol and 5% v/v glycerol).

Fractions were collected by washing the loaded fractions with buffer B. The fractions

corresponding to the AAG peak were then analyzed by SDS-PAGE and the AAG

containing fractions were pooled and concentrated as before to approximately 1.5 ml

final volume. The purified, concentrated AAG was then dialysed against two changes of

pre- chilled storage buffer( 50mM HEPES, pH 8.0, 100mM KC1, ImM EDTA, ImM

dithiothreitol and 37.5% v/v glycerol), in a 12, 500 cut-off dialysis membrane, for atleast

4 hours each. The final protein yield was determined by A280 measurements against a

storage buffer blank using the published AAG extinction co-efficient of

(27, 099 M^cm-1). The dialyzed, pure AAG was dispensed into pre- chilled eppendorf

tubes as 8- 10L aliquots and stored at 800C. The purity of the protein was assessed by

staining a SDS- PAGE gel with sypro-red dye.

Typically, fresh aliquots were taken from the -800C for an experiment. Any protein

leftover from the used stock was stored at -200C and used within 3 days of storage. Serial

dilutions of the stocks were done in cold AAG storage buffer prior to an experiment.

Synthesis and Purification of Oligonucelotides

Synthetic oligonucleotides were made on an ABI 392 DNA synthesizer using

standard P-cyanoethylphosphoramidite chemistry and reagents from Glen Research

(Sterling, VA). The oligonucleotides were cleaved after synthesis from the solid support

by treatment with fresh ammonium hydroxide and were forced into collection vials with

argon flush. The collected oligonucleotides contained base-protected products and other

partial synthesis products. The protecting groups were removed by placing the vial into a










55C sand bath for 12-16 hours. The deprotected oligos were then concentrated to

dryness in a Savant Hi-Speed centrifugal vacuum system. The dried oligonucleotides

were resuspended in 50% glycerol for purification by denaturing PAGE.

The samples were then resolved on a 12% polyacrylamide gel containing 8M urea,

by loading around 40- 60 nM DNA per lane. The bands corresponding to the right sized

product were visualized by UV shadowing and cut out from the gel. The DNA was eluted

by shaking the gel slices in 8-15 mL of sterile NTE buffer ( 50mM Tris, pH 7.5, 50mM

NaCl and ImM EDTA), at room temperature. The buffer was changed every 8 hours

three times and the eluted DNA was pooled. The pooled DNA was then passed through a

0.2-[tm syringe filters (Acrodisc) to remove the gel remnants before dialyzing in a 3500

MW cut-off dialysis tubing (Spectropor) against 4L of nanopure water, changing water

every 8 hours, 3 times. The dialyzed DNA was concentrated to approximately 1.5mL

final volume and then quantitated using A260 measurements and calculated extinction co-

efficients (94, 114). The oligonucleotides were then aliquoted and stored at -800C.

Radio-labeling of Substrates and Annealing to Complement

DNA substrates strands containing the damaged nucleotide were 5'end-labeled

with [y]32P-ATP using T4-kinase. A typical 50-[tL reaction consisted of 2[tM single-

stranded 25mer containing the damaged base, labeling buffer (50mM HEPES, pH 8.0,

100mM KC1, 6.4mM MgCl2), 30[tCi [y]32P-ATP (Amersham) and 1 unit of T4 kinase

(Invitrogen) at 370C for at least one hour. The kinase was then inactivated by placing the

tube at 950C for 5 minutes. A 2-fold excess of the unlabeled complement was added to

ensure complete annealing of all the labeled substrate. Annealing was performed by

thoroughly heating the mixture to 850C to remove all secondary structure and then slowly









cooling to room temperature to aid the formation of the most stable duplex. Typically

10% of the total DNA substrate was labeled when using a final DNA concentration of

2[LM. When a final concentration of 200nM substrate was used, with all other parameters

remaining constant typically almost 100% of the substrate was labeled. For freshly

labeled substrate, optimum times of exposure for good signal ranged from 6-8 hours.

Increasing times of exposure were used for older labels, ranging from 10-16 hours.

Single Turnover Excision Assay for Glycosylase Activity

To provide a measure of the chemistry of the enzyme, single turnover conditions in

which a known amount of substrate DNA was incubated with a vast excess of AAG, were

used. Single turnover conditions provide a measure of a step occurring after substrate

binding and before product dissociation, which in this case is presumed to be the

chemistry step. Another reason for doing single turnover kinetics for assessing activity is

that, in preliminary steady- state experiments, it was observed that AAG could not

recycle to excise all the substrate present and hence seemed to be product inhibited in

some way. Saturating conditions were used to assess glycosylase activity. Typically,

either 50nM labeled duplex DNA (10% labeled) or 5nM labeled duplex DNA (100%

labeled) were used as substrate in the AAG assay buffer, consisting of 50mM HEPES pH

8.0, 100mM KC1, 0.5mM EDTA, 0.25mM DTT and 9.5 % v/v glycerol. At 370C, the

assay was started by adding AAG, typically anywhere from 4-to 20-fold excess AAG

over substrate was used depending on the saturating concentration for that particular

substrate, which were determined empirically by titrations. At various time points, from

30 seconds to 80 minutes, a 4[LL aliquot of the reaction was withdrawn and quenched in

0.2M NaOH and chilled to stop the glycosylase activity. The rest of the assay relies on









the fact that the abasic site after glycosylase action exists in equilibrium between the

hemiacetal and the aldehyde forms of the deoxyribose. The aldehyde form is labile to

base and heat and undergoes p-elimination to yield a single stranded 5' labeled product

upon denaturation, indicative of AAG mediated excision of the substrate base. After

quenching and chilling, the tubes were heated at 950C for 10-15 minutes to ensure

complete P-elimination. The samples are then diluted with 2 volumes of loading buffer

consisting of 95% formamide and 20 mM EDTA. Unreacted substrates were separated

from cleaved products by PAGE on 12 % denaturing gels (8M urea, 1XTBE). The dried

gel was exposed to a phosphor screen and visualized using a phosphorimager (Storm,

Molecular Dynamics). Only the DNA with the 5'-label is visible under these conditions,

resolved between a faster migrating 13mer product of AAG activity and a slower

migrating, unexcised 25mer substrate. The bands were then analyzed using Image Quant

software, which enables to quantify the P-emissions. The number of counts is

proportional to the amount of DNA in the band. The background radioactivity was

controlled for by averaging the analyzed bands with the counts in region of the gel which

has no DNA. The product formed over time Pt was plotted over time using Kaleidagraph

software. The plot was fit to a single- exponential rise Equation- 3-1, using non- linear

regression.

Pt Ao(1- e-kobs) (Eqn 3-1)

Where, Ao and kobs are the amplitude of product formed and observed rate of the

exponential rise, respectively.










5' *

3'
=32P label

370C hAAG

5' *

X i T I T i i i i I I


950C 0.2M NaOH




3' T T,,1 1
EE E E EEEE&
m m m m m m m m m m m m


PAGE Analysis of Reaction Products


00a a o -


5' l ii i1 6 6 6 6 6 6 6


.. 5*nn r-r

0 2 4 8 20 40 80

Time (min)


Figure 3-2. A schematic of the excision assay and a sample gel for resolution of products
from substrates. The glecosylase action, followed by base and heat exposure,
causes complete p-elimination to yield a 13-mer product, which is resolved on
the gel from the 25-mer substrate. The 0 min time point is the no AAG control
which also serves as the background control for spontaneous generation of
basic sites during the assay.

Multiple Turnover Assays for Glycosylase Activity

Under multiple turnover conditions, concentrations of DNA (50nM) was in excess

of AAG (1-20nM) were used. Other assay conditions and procedures were the same as

for the single turnover assays described above. To ensure total denaturation of the DNA

after heating, twice the volume of Formamide/EDTA was used..

Electrophoretic Mobility Shift Assay (EMSA) for AAG binding activity

Concentrations of labeled duplex DNA used to observe the binding properties of

AAG were the same as used in the excision assays. Because, AAG was capable of









excising the damaged base, a catalytically inactive mutant of AAG, E125Q, in which the

catalytic residue Glu-125 was mutated to a Gln was used. This mutant has identical

binding properties to the wild type AAG, but cannot excise the glycosidic bond. The

EMSA consists of resolving bound versus free species on a non- denaturing PAGE, to

give a higher molecular weight, slower migrating bound species and free, faster migrating

species. Ideally, 50nM DNA was incubated on ice with 0 to 1600nM E125Q for 10

minutes and loaded on to a 6% non-denaturing PAGE, which was then run at 8V/cm for

180 minutes at 40C, to prevent overheating of the fragile gel. When 5nM DNA was used,

0 to 160 nM E125Q was used. The dried gels were analyzed by phosphorimaging and

ImageQuant software as in the excision assays to obtain free over bound DNA. DNA

bound was plotted over concentration ofE125Q and fit to a quadratic equation (Equation

3- 2) to get apparent binding constants (Kd)


E, +D, +Kd J(E, +D, +Kd )2 + 4E,D,
[EDtotai] = (Eqn 3-2)

Melting Temperature (Tm) Measurements For Duplex DNA

Melting temperatures of duplex DNA substrates were calculated using the Thermal

program for DNA melting in the CARY-3 Bio-UV-visible spectrophotometer (Varian

Australia Pty Ltd., Australia). A temperature controller (Peltier) was used to create a

controlled rise in temperature. DNA substrates containing the damaged base were

annealed to equal concentrations of the complementary strand containing T, F or C

opposite the damaged base in 50mM HEPES pH 8.0, 100mM KCl and 0.5mM EDTA, to

a final duplex concentration of 4[LM. Annealed DNA was diluted 6 times into masked

cuvettes with caps to prevent evaporation during melting. The dilution gave a final

concentration of 0.67[LM, which was determined to be the minimum concentration









required to give an initial absorbance of 0.2, so as to be within the range of the Beer-

Lambert law of absorbance. The temperature was increased from 350C to 650C at a rate

of 0.50C per minute. Tm values were calculated by taking the first derivative of the

melting curve. Tm values correspond to the maximum value of the first derivative.

Control experiments were done using temperature increase rates of 0.25, 0.5 and 1C per

minute. Rates of 0.25 and 0.50C per minute gave consistent results.

Fluorescence Assay For Ethenoadenine-AAG Binding and Excision

gA is intrinsically fluorescent(94) and has an excitation Xmax at 3 10nm with an

emission Xmax at 405nm (Figure 3-3). The fluorescent properties of sA were used to

monitor the binding and excision of AAG in real time. The fluorescence emission of sA

is considerably quenched when in double stranded DNA, but upon binding and excision

of the glycosylic bond by AAG, fluorescence emission is increased in intensity in a time

based pattern indicative of AAG activity.

Duplex DNA (100nM) containing sA as the damaged base was incubated with

saturating enzyme concentrations. For measuring binding in the absence of excision, 400-

1600nM E125Q was used in standard AAG buffer, in which 0.5M HEPES was replaced

by 0.5M Bicine(pH 8.0) as HEPES was found to be slightly fluorescent at the UV range.

Replacing HEPES with Bicine did not affect AAG activity, as measured in 32P

glycosylase assays. Binding reactions were carried at 250C. To observe excision, AAG

was used in the same concentrations as in the binding measurements and the reaction was

carried out at 370C. EA fluorescence was monitored over time in a quartz cuvette. Data

was collected using a Photon Technology Inc. QuantaMaster fluorimeter using a 75W

xenon- arc lamp. The band pass was set at 4nm with the excitation and emission










monochromators set at 310nm and 410nm, respectively. Although the theoretical Xmax for

emission was found to be 405nm, reactions were monitored at 410nm to avoid the

tryptophan fluorescence interference due to the protein at this wavelength. Each

concentration titration was done as a separate reaction. Buffer only and DNA only

background signals were recorded for all spectra, before adding the enzyme and starting

the reaction.



70000

60000 -

560000 -

40000 -

o0000 -
20000 -



10000 -

0 ----- i --- i --- i --- i --- i ---
200 250 300 350 400 450 600 56C
VWvelength (nm)

Figure 3-3. Fluorescent properties of 100nMsA when in double stranded DNA and after
excision by 400nM AAG at 370C for 60 minutes. The blue and green spectra
are excitation spectra, before and after excision by AAG, while the red and
brown spectra are emission spectra, before and after excision b y AAG. The
xmax for excitation and emission are given by the peaks (see text).

Stopped-flow Fluorescence to Observe Flipping of EA by AAG

Since binding appeared to be too fast to measure by hand- mixing in the cuvette,

stopped-flow measurements were done to observe binding and flipping in real time. DNA

containing sA, at a final concentration of 200nM, was added to AAG at a final









concentration of 100, 200 or 400nM in standard AAG buffer at 200C in a Biologic

Stopped-flow fluorimeter. Excitation was set at 3 10nm and emission was measured in the

2-channel mode using 380nm cut on filters. Traces were taken for each reaction with 4

traces per channel, giving a total of 8 traces per mixing. The same number of traces was

taken for the DNA only and AAG only controls for subtracting the backgrounds.

Maximum number of traces was taken to increase the signal to noise ratio as much as

possible. These traces were then averaged to obtain the final signal, which was plotted

against time to obtain a 5000millisecond time based binding curve. The data was not

fitted, but the change in fluorescence intensity was taken as a measure of binding and

flipping of sA.














CHAPTER 4
EFFECTS OF HYDROGEN BONDING WITHIN A DAMAGED BASE PAIR ON THE
ACTIVITY OF WILD-TYPE AND DNA-INTERCALATING MUTANTS OF HUMAN
ALKYLADENINE DNA GLYCOSYLASE

Structural studies of AAG (95, 96) and other DNA glycosylases have revealed that

a nucleotide "flipping" mechanism is used for damaged base recognition and excision in

which the damaged base is flipped out of the DNA helix and bound in an enzyme active

site. In these nucleotide-flipped DNA glycosylase*DNA complexes, an enzyme amino

acid side chain is inserted into the base stack at the site vacated by the flipped base and

may assist in nucleotide flipping by pushing the damaged base from the helix. It is

believed that DNA glycosylases actively flip damaged bases out of the helix rather than

passively capturing bases that have transiently adopted extrahelical conformations. This

active nucleotide flipping mechanism is supported by detailed kinetic studies ofE. coli

uracil DNA glycosylase which show a two-step binding mechanism where UDG initially

binds DNA to form a non-flipped protein-DNA complex prior to flipping uracil from the

helix (97).

Many questions remain about how nucleotide flipping enables DNA glycosylases

to discriminate between damaged and undamaged bases. For DNA glycosylases that

have a narrow substrate specificity, a mechanism in which a "tight fit" of the damaged

base in the enzyme active site allows the DNA glycosylase to discriminate between

damaged and undamaged bases seems probable. For example, UDG excises only uracil

from DNA and mutation of enzyme residues that form specific interactions with U alters

the specificity of the enzyme so that it can excise C and T (96, 95). On the other hand,









for DNA glycosylases that excise a structurally diverse group of damaged bases such as

AAG, a mechanism for damaged base recognition and excision that depends solely on

specific interactions between enzyme binding pocket residues and a damaged base seems

unlikely. Damaged bases excised, by AAG including 3-methyladenine, 1,N6-

ethenoadenine (EA), hypoxanthine (Hx), and 7-methylguanine, have no obvious structural

features in common that would allow the enzyme to distinguish between damaged and

undamaged bases. In addition, the efficiency of excision by AAG is dependent on the

base pairing partner for some damaged bases (76) even though the enzyme makes no

specific contacts with the base pairing partner in the crystal structures (77, 78). This base

pair specificity of AAG further suggests that substrate specificity is governed by a

mechanism that involves more than the fit of the damaged base in the enzyme binding

pocket.

To further define the mechanisms of damaged base recognition and excision by

AAG, the question of how nucleotide flipping contributes to the efficiency of base

excision by AAG was addressed using two general approaches. First, site-directed

mutations that were predicted to reduce the efficiency of nucleotide flipping were made

to the DNA intercalating Tyr-162 residue of AAG. Second, hydrogen bonding

interactions within the damaged base pair were removed by substitution of a non-

hydrogen bonding partner, difluorotoluene (F), for thymine to increase the efficiency of

nucleotide flipping by reducing the stability of the damaged base within the helix.

Difluorotoluene is isoteric to thymine but lacks the hydrogen bonding potential due to the

substitution of the hydrogen bonding groups of thymine with electronegative fluorine

(Figure 4-1). Kool and co- workers who designed F as a thymine analog found that it was









a substrate of replicative polymerases and can be inserted into a growing strand just like

thymine, making it a valuable tool for use in replication and repair studies (105, 106).

Since AAG substrates are damaged adenine bases encountered opposite T, F is a good

reagent to use as a non- hydrogen bonding partner as it was a thymine analog.

Experiments were designed to investigate the effect of the Tyr-162 mutation on AAG

activity and the effects of the "DNA mutation" in which T was replaced with F, on the

activities of both wild type and mutant AAG.

.H3 F .H.
Ov "-*HN NH N O H
HN / N-H"""0 HN N-H F



Hx-T Hx*F




HN N H- FH
gP_ NH HN/ r -J H-
F

SA*T 6A*F




Figure4-1. Chemical structures of hypoxanthine and 1, N6-ethenoadenine paired with
thymine and difluorotoluene. F cannot form hydrogen bonds with Hx. Neither
T nor F cannot hydrogen bond with EA.

DNA Substrates and Sequences

The two AAG substrates used in these experiments were Hx and EA. DNA

duplexes were 25 nucleotides long, with the damaged bases in position 13, base paired

with either T or F, with the rest of the sequence remaining perfectly complementary. The









DNA substrates were always duplexes, since AAG was found to be incapable of binding

or excision on single stranded DNA. The substrate DNAs were named based on the

central damaged base and its base pairing partner, as Hx*T, Hx*F, A*oT or sA*F. The

sequences of the DNA substrates are tabulated in Table 4-1.

Table 4-1. Sequences of DNA substrates and positions of damaged base pairs
Upper 5'-GCG TCA AAA TGT NGG TAT TTC CAT G-3' (N= Hx or sA)
strand
Lower 5'-CAT GGA AAT ACC XAC ATT TTG ACG C-5' (X= T or F)
strand


Mutations to Tyr-162 and Projected Consequences

To assess the contribution of the DNA-intercalating Tyr-162 residue to the base

excision activity of AAG, Tyr-162 was converted to Ser and Phe by site-directed

mutagenesis to generate two mutant proteins, Y162S and Y162F, respectively. A

catalytically inactive double mutant, Y162F/E125Q, was made for DNA binding

experiments. Converting the Tyr-162 residue to Ser removes the aromatic ring generating

a smaller amino acid side chain that should not be able to penetrate the DNA helix as

deeply when intercalated. Mutation of Tyr-162 to Phe removes the hydroxyl group but

leaves the aromatic ring intact to intercalate into the DNA base stack. These differences

in insertion are illustrated in Figure 4-2 as cartoons in which Tyr-162 is replaced by Ser

or Phe, as seen in the crystal structure. Figure 4-2 is not an actual structure but is meant to

indicate possible differences in intercalation between Tyr-162 and the two amino-acids

by simple replacement and not to indicate any other properties they may affect such as

flipping. To see whether these intercalating differences will be translated into flipping

inefficiencies is the goal of this mutational analysis.









wt
Tyr-162 Ser-162 Phe-162










.I.Y




Figure4-2. Projected differences in intercalation ability of Ser-162 and Phe-162 when
compared to the wild type residue, Tyr-162, based on the crystal structure of
AAG bound to EA. Shown is a close- up view of the P-hairpin (blue ribbons)
and the intercalating residue (red space-fill) positioned in the helix (green
ball and sticks) to fill the space vacated by the flipped- out EA (yellow ball
and sticks). Ser and Phe may have different abilities to intercalate than Tyr
(see text).

Base Excision and DNA Binding Activities of the Y162S Mutant

Base excision activity for the Y162S mutant was measured in a chemical

cleavage/gel assay for DNA substrates using saturating AAG concentrations, in which

excision of all the substrate is expected based on previous kinetic studies. The strand

containing the damage was end-labeled with 32p, prior to annealing to its complementary

strand to create duplexes of otherwise identical sequences that contained Hx*T and sA*T

base pairs. In 60minute assays using 1600 nM Y162S and 50 nM DNA substrate, no

detectable base excision was observed for either DNA substrate. We estimate that the

Y162S mutant is at least 1000-fold less active than the wild type AAG enzyme based on

this result and using the conservative assumption that 1 nM product (2% reaction) would

have been detected if formed in these assays.










The DNA binding activity of the Y162S mutant was measured in electrophoretic

mobility shift assays (EMSA) with the same damaged duplexes as used in excision

assays, where the damage-containing DNA strand was 5'-end-labeled with 32P. A

damage-specific protein-DNA complex was not observed for the Y162S mutant with

DNA substrates containing Hx or sA opposite T (Figure 4-3). At high Y162S

concentrations in the EMSA, a general smearing of the DNA band was observed in a

pattern similar to that for AAG with undamaged DNA (not shown). This smearing may

represent weaker damage-independent DNA binding.



EA-T Hx*T


Free DNA-+ 5U a
800 800
[Enzyme]nM-- 0 0

Figure4-3. Electrophoretic mobility shift assays to measure the affinity of the Y162S
mutant for DNA containing an sA*T or a Hx*T base pair. 50nM labeled,
duplex DNA was incubated with 0- 800nM Y162S mutant. A band
corresponding to a damage-specific protein-DNA complex is not observed for
the Y162S mutant in assays with either an sA*T pair (left panel) or an Hx*T
pair (right panel). Smearing of the free DNA band is observed at 400 and 800
nM Y162S and may represent weaker damage-independent DNA binding.









Activity of the Y162F Mutant

Base Excision by the Y162F Mutant

Single turnover kinetics of excision of Hx when paired with T was measured in a

chemical cleavage/gel assay for both AAG and the Y162F mutant. Enzymes, at

concentrations of 400, 800, and 1600 nM, in two separate experiments at each

concentration, were incubated with 50 nM 32P-labeled 25-nt duplex DNA substrates at

370 C. Aliquots of each reaction mixture were withdrawn at several time points,

quenched, and analyzed by PAGE to quantitate the concentration of products formed.

For each enzyme, reaction time courses were essentially the same at all three

concentrations demonstrating that single-turnover conditions were met. Individual time

courses were fit empirically to an exponential rise to calculate observed rates (kob).

Average values and standard deviations for kobs calculated from all six experiments (two

at each enzyme concentration) are shown in Table 4-2. Excision of Hx was 4-fold more

rapid in assays with AAG than the Y162F mutant. The reaction course for 400nM

enzyme is shown in Figure 4-4.

Because AAG catalyzes excision of a structurally diverse group of damaged

purine bases, the possibility that the Y162F mutation may have differential effects on

excision of different damaged bases was tested. Kinetics of excision of the structurally

dissimilar 1,N6-ethenoadenine placed opposite T were measured in single-turnover assays

containing 400 and 800 nM enzyme. Reaction courses are shown in Figure 4-5. For each

enzyme, observed rates were the same at both enzyme concentrations. The Y162F

mutation had a smaller effect on the single turnover excision rate for sA where AAG was

1.7-fold faster than the Y162F mutant (Table 4-2).










To determine whether rates of excision of Hx would increase by making the base

easier to displace from the helix, the T opposite Hx was replaced by difluorotoluene (F),

which does not form hydrogen bonds with Hx (Figure 4-1). Single-turnover kinetics of

excision of Hx opposite F was measured in the chemical cleavage/gel assay with 50 nM

DNA and 400, 800, and 1600 nM enzyme (Table 4-2). Excision rates were not

dependent on enzyme concentration for either AAG or Y162F. Excision activities for

both AAG and the Y162F mutant increased on the Hx*F DNA substrate relative to the

Hx*T duplex (data for 400 nM enzyme are shown in Figure 4-4). The magnitude of the

increase was greater for the Y162F mutant (3.5-fold) than for AAG (2-fold).

It is possible that the increased excision activity could be due to some effect of

replacing T with F other than removing hydrogen bonding interactions. To rule out this

possibility, excision was also measured for DNA substrates containing sA*T and EA*F

base pairs. gA does not form Watson-Crick-type hydrogen bonding interactions with

either T or F. Single-turnover kinetics of excision of sA opposite F were measured in

chemical cleavage/gel assays using 50 nM DNA and 400 and 800 nM enzyme in separate

experiments (Table 4-2). There was not a significant effect on excision rates of sA, as a

1.2-fold decrease in the excision rate for AAG and a 1.1-fold increase for the Y162F

mutant were observed (Figure 4-5).













50 I, , I 50 ,


40O 40


I30 7 30


0 20 2, 20

400nM AAG M 400nM Y162F
: 10 10
-- 50 -M Hx-T -*-50 nM Hx*T
--50 nM HxF m- -50 nM Hx-F

01 . .I I. I .
0 5 10 15 20 0 8 16 24 32 40 48 56 64
Time (min) Time (min)




Figure 4-4. Plots of time courses for Hx excision by wt AAGA79 and Y162F. Plots of
the concentrations of abasic DNA product formed as a function of time in
assays containing 400 nM enzyme and 50 nM DNA are shown. Because there
is a relatively large difference in rates of Hx excision, time courses for
excision of Hx paired with T (triangles) and F (squares) are plotted in
separate graphs on different time scales for AAG(left) and theY162F mutant
(right). Data plotted are average values from two independent experiments
with standard deviations. Solid lines are the result of a single exponential fit
to the data.









50


40
1 800nM T
wtAAG -



30
Ch 800nM
SPhe mutant
x
4) 20




-- 50nM EA*F


0 8 16 24 32 40 48 56 64

Time (min)
Figure 4-5.Plots of time courses for sA excision by wt AAGA79 and Y162F The base
pairing partner, T or F, did not affect EA excision rates for either enzyme as
demonstrated by overlapping time courses for excision of sA by AAG on
sA*T (green squares) and EA*F (blue squares) DNA and Y162F on sA*T (red
diamonds) and EA*F (orange diamonds) DNA. Data plotted are average
values from two independent experiments with standard deviations. Solid
lines are the result of a single exponential fit to the data.

Results obtained from removing hydrogen bonding interactions to Hx indicated

enhanced rates of excision of Hx by both the wt and the Y162F mutant. Normal purines

are not usually excised by AAG. Experiments were done to see if any enhanced excision

of normal purines opposite F was seen. In control experiments in which DNA substrates

containing no damaged base, but adenine or guanine opposite F (A/G*F), it was seen that

removing hydrogen bonding interactions did not make these normal DNA bases

substrates for AAG. Similar AAG assays were performed to confirm this fact (Figure 4-

6). Some groups have observed excision of normal bases by AAG at low levels, but we

have not been able to demonstrate this activity in the presence or the absence of hydrogen









bonding interactions. The observations of these groups may be due to different exposure

times of the gels or very long assay times. The non- excision of normal purines opposite

F goes on to further prove the fact that AAG uses flipping as an essential step in

recognition but also co- ordinates flipping with the active site parameters that decide

what a substrate is. So though A or G could be made easier to flip by putting opposite F,

excision is not observed due to active site constraints.

Table 4-2. Observed excision rates and relative activities of AAG and Y162F mutant
Enzyme Base pair kobs min-la krel
(AAG/Y 162F)b
AAG Hx*T 0.62+0.19 4.1

Hx*F 1.30.1 2.5
EsAT 0.0620.003 1.7
EsAF 0.0520.005 1.2
Y162F Hx*T 0.150.03

Hx*F 0.530.13
EsAT 0.0370.001
EsAF 0.0420.002


a,Values for kobs were calculated from single exponential fits to individual experiments.
For Hx base pairs, two independent experiments were done at enzyme concentrations of
400, 800, and 1600 nM and average kobs values and standard deviations are reported for
all six experiments. For sA pairs, two independent experiments were done at 400 and 800
nM enzyme and average kobs values and standard deviations are reported for the four
experiments. b, Relative values, krel, were calculated from the ratio of the kobs for wt to
Y162F for each base pair.












50nM A*F 50nM G*F


C 20 40 60 80 100 Time(min) C 20 40 60 80 100

25 mer --D I











Figure 4-6. Control glycosylase assay to show that base pairing with F does not make A
or G excisable by AAG. 100 minute time course gels are shown, with 50nM
A*F and G*F DNA duplexes incubated with 800nM AAG at 370C and
quenched at the indicated time points. The C lane is the enzyme- less control
reaction. Only 25-mer substrates are seen, no 12-mer product, indicative of
excision by AAG is seen on the gel.

DNA Binding Ability of Y162F Mutant

Mutation of the Tyr-162 residue to Phe reduces the DNA binding activity measured

in electrophoretic mobility shift assays (EMSA). For and EMSA, 50 nM 32P-labeled

duplex DNA substrates, identical to those used in excision assays, were incubated with

increasing concentrations of enzyme (10 800 nM) prior to nondenaturing PAGE

analysis. Since base excision would convert DNA substrates to products during the time

course of EMSAs, catalytically inactive mutants (E125Q) of AAG and Y162F were used

in these assays. The affinity of the Y162F/E125Q mutant for DNA containing a Hx*T

pair is reduced relative to E125Q (Figure 4-7, upper panels). A concentration of 50 nM

Y162F/E125Q is needed to form a similar fraction of enzyme.DNA complex as seen with









20 nM E125Q. At concentrations of 400 nM enzyme, about 70% of the DNA is bound

by E125Q whereas about 25% is bound by Y162F based on the intensity of the bands. As

reported previously (100), AAG binds to DNA containing an sA*T pair with greater

affinity than a Hx*T pair (Figure 4-7 and Figure 4-8, upper left panels). This is also true

for the Y162F/E125Q mutant (Figure 4-7 and Figure 4-8, upper right panels). The

Y162F/E125Q mutant binds DNA containing an sA*T pair more weakly than E125Q as it

takes 20 nM Y162F/E125Q to form about the same concentration of enzyme-DNA

complex as 10 nM E125Q.

To determine what effect substitution of T with F would have on the DNA binding

activity of AAG, assays were done for DNA substrates containing Hx*F and sA*F pairs.

Binding assays contained 50 nM 32P-labeled duplex DNA and increasing concentrations

of AAG E125Q or Y162F/E125Q (10 800 nM). For both enzymes, binding was similar

for DNA duplexes containing Hx*T and Hx*F pairs (Figure 4-7, lower panels), and

binding was slightly enhanced on duplexes containing sA*F in comparison with sA*T

(Figures 4-8, lower panels).









E125Q


Enzyme-DNA
complex


Free DNA -
[Enzyme] nM


Hx*T


a v-


c00000000
WWA ^ B


Y162F/E125Q


Hx*T


00000000
r I 0000
rNqin


Hx*F


Hx-F


Enzyme-DNA
complex -*


-MIA


Wi


Free DNA--
[Enzyme] nM 'nooo
M- WN CO


N0000C 0
r -LI D O0 O


Figure 4-7. Binding of wt AAGA79 and the Y162F mutant to DNA containing Hx*T and
Hx*F base pairs. Assays were done with duplexes containing either a Hx*T
(upper panels) or Hx*F (lower (nels) pair. Increasing concentrations (10 to
800 nM) of E125Q (left panels) and the E125Q/Y162F mutant (right panels)
were incubated with 50 nM DNA. Both enzymes were catalytically inactive
mutants of A79AAG and the Y162F mutant, respectively. They were used so
that binding efficiency can be observed in the absence of excision. Binding
constants were not obtained due to the high degree of smearing which
hampered exact quantitation.


b" baw



b" baw










E125Q


Y162F/E125Q


EA-T


Enzyme-DNA
complex -


Free DNA- -l-
[Enzyme]nM o o o 8o o o
-'-- -V- CM0 W00


eA*F


00000000
N .3 D 0000
'-CI tan


sA-F


iI


Enzyme-DNA
complex


Free DNA--* iW .
[Enzyme]nM o oc3 0o 0 0
rN too)


Figure 4-8. Binding of AAG and the Y162F mutant to DNA containing sA*T and sA*F
base pairs. EMSA assays were done as in Figure 4.6 with 25-nt duplexes
containing either an sA*T (upper panels) or sA*F (lower panels) pair at
position 13. Increasing concentrations (10 to 800 nM) of wt AAG (left
panels) and the Y162F mutant (right panels) were incubated with 50 nM
DNA. Catalytically inactive mutants are used as in Figure 4.7.
Implications of Flipping in the Catalytic Efficiency of AAG
The ability of DNA glycosylases to identify and excise damaged DNA bases is key
to the overall success of base excision repair. Structural studies of AAG reveal that


0O00000 0
S CML 0 0 0 0
r(%l aO


sAT









flipping of the damaged nucleotide is the first step in substrate recognition, and this is

facilitated by intercalating Tyr-162 into the space vacated by the flipped out nucleotide.

In this study, the DNA-intercalating Tyr-162 residue of AAG was converted to

serine (Y162S) and phenylalanine (Y162F) by site-directed mutagenesis. A decrease in

the base excision activities of both mutants was observed as expected if the Tyr-162

residue contributed to nucleotide flipping by helping to push the damaged base from the

helix. Base excision and damage- specific binding activities of the Y162S mutant were

reduced to undetectable levels for DNA substrates containing Hx*T and sA*T pairs,

indicating that this mutant must be at least 1000-fold less active than AAG. The fact that

DNA binding activity of the Y162S mutant was not detectable by EMSA suggests that

the enzyme-DNA complex seen for AAG is a nucleotide flipped complex.

A similar mutation in UDG converting the DNA-intercalating Leu residue to Ala

resulted in an 8 to 80-fold decrease in excision activity and mutation of Leu to Gly

reduced UDG's excision activity by a factor of 100 600 (91, 92). The comparatively

large effect of the Y162S mutation on AAG's activity may reflect a greater contribution

of the DNA-intercalating residue to the activity of AAG than UDG.

Mutation of Tyr-162 to Phe leaves the aromatic ring to intercalate in DNA but

removes the hydroxyl group from the aromatic ring. This mutation decreases the size of

the DNA-intercalating residue much less than the Ser mutation but still affects the

excision activity of the enzyme. Excision of Hx when paired with T by the Y162F

mutant is 4 times slower than excision by the AAG and excision of sA paired with T is

1.7 times slower. Interestingly, the activity of the Y162F mutant is "rescued" on a DNA

substrate where Hx is paired with F. The excision rate for the Y162F mutant increases to









the rate measured for AAG excision of Hx paired with T. It is possible that making Hx

easier to flip in the context of an Hx*F pair counterbalances a deficiency in the flipping

ability of the Y162F mutant. An alternative explanation for the effect of the Y162F

mutation on the excision activity of AAG is that the slightly smaller Phe residue is not

able to "push" the displaced base as far into the enzyme binding pocket to align it

properly for catalysis. If this were true then no difference in excision rates for Hx when

paired with T and F would have been seen because the Phe mutant would have "pushed"

Hx the same distance in both cases.

The rationale for replacing T with F in Hx base pairs was that F is isosteric with T

having the same overall shape but will not form hydrogen bonds with Hx. The

expectation was that the lack of hydrogen bonding will increase the ease of flipping Hx

by decreasing the stability of the base pair. To rule out the possibility that F could have

some other unanticipated effect on excision activity, excision of sA was measured when

paired with T and F where neither pair forms hydrogen bonding interactions.

Substitution of T with F had no significant effect on excision rates of sA for either AAG

or the Y162F mutant whereas it increased the excision rate of Hx by a factor of 2 for

AAG and about 3 to 4 for the Y162F mutant. Thus, the increase in Hx excision rates is

likely to be due to changes in hydrogen bonding interactions in the Hx pair. These results

are consistent with a model where the ease of flipping a damaged base contributes to the

base pair specificity of AAG.

The kinetic mechanism for base excision by AAG is likely to contain a nucleotide

flipping step in addition to the chemistry step where base excision occurs. Changing the

ease of nucleotide flipping either by mutations to the enzyme or by changes to the









stability of a damaged base within the helix affects single turnover excision rates by

altering the population of substrates stably flipped. The observation that substitution of T

with F increases single turnover excision rates of Hx for both AAG and the Y162F

mutant suggests that nucleotide flipping is important for Hx excision. An altered flipping

equilibrium for Hx excision would also explain the base pair specificity observed

previously (75, 76). Two explanations are possible to explain why excision of sA is not

affected to a great degree by its base pairing partner. Either the nucleotide flipping step

may not alter the flipping equilibrium as drastically as for Hx, or the flipping equilibrium

is not affected by the base pairing partner since sA lacks hydrogen bonding interactions

with its partner.

A Two-step Selection Model for AAG Activity

Based on these results that were published (75) and previous work, we have

developed a working model that explains the damaged base and base pair specificity of

AAG. We propose that the specificity of base excision by AAG is governed by two

important selection steps, nucleotide flipping and chemistry of bond cleavage which is

affected by many factors such as proper fit of the base, alignment of the bond, suitability

of the leaving group etc. in the enzyme active site. The enzyme may use the ease of

flipping a damaged base as the initial criterion for discriminating between damaged and

undamaged bases and then use fit of the damaged base in the active site as a final check.

The first nucleotide flipping selection step would be affected by changes in local DNA

sequence or structure that affect the stability of a damaged base within the helix. Once a

damaged base is flipped, it still must be aligned properly in the active site for hydrolysis

of the glycosidic bond to occur. This second criterion, proper fit in the active site, would

explain why Hx but not G is excised from a wobble-type base pair with T (76). The 2-









amino group may prevent G from fitting in the active site properly (78). The other factors

in the active site may include suitability of the leaving group for chemistry, alignment of

the glycosidic bond relative to the activated water and so on. An implication of this two

step selection is that the overall efficiency of base excision repair may be a function of

local DNA sequence and structure which affect the stability of damaged bases in the

helix. A dependence on the efficiency of base excision on DNA sequence and structure

could contribute to the formation of mutational "hot spots" and "cold spots". Both AAG

DNA-intercalating mutants and the difluorotoluene base pairing partner will be useful

tools for testing this model further.














CHAPTER 5
ACTIVITY OF HUMAN ALKYLADENINE DNA GLYCOSYLASE IS SENSITIVE
TO THE LOCAL SEQUENCE CONTEXT OF THE DAMAGED BASE

The contribution of nucleotide flipping to the excision efficiency of DNA

glycosylases is not yet fully understood. Especially with respect to a glycosylase like

AAG, which has a diverse substrate range, the contribution of flipping could be critical to

how the enzyme discriminates between substrates and non- substrates and also how it can

accommodate a diverse group of substrates. The contribution of flipping to substrate

specificity could be unique for AAG since many other glycosylases such as UDG act on a

single substrate, in this instance, uracil only. But AAG acts on substrates ranging from 3-

methyladenine and hypoxanthine (Hx) to 1, N6- ethenoadenine (EA), which are not

structurally related enough to enable the enzyme to use a common mechanism of

recognition. Though the crystal structure of AAG does not indicate any specific contacts

between the enzyme and the base pairing partner to the flipped out damaged base, it has

been shown by us that the excision efficiency of some damaged bases is dependent on the

identity of the base pair and not the base alone (75, 76). The difference in excision can be

directly related to the ease of flipping the damaged base in a given base pair because,

removing hydrogen bonds which can make flipping easier, also increased excision

efficiency, in the case of Hx (75). This indicated that stability in DNA could affect repair

of Hx by AAG. The minimum two- step mechanism for recognition and binding to the

substrate discussed in the end of chapter 4 served as an additional guide to come up with

novel methods to understand the contribution of flipping to Hx excision. The first step









was to identify additional factors around Hx that could contribute to its flippability by

AAG. Since base stacking in DNA is a major stabilizing source, changes to the flanking

neighbors to Hx were made, using either relatively weak or strong base stacking partners.

These may either further stabilize or destabilize the Hx base pair in DNA and thus affect

its flipping by AAG. To the base stacking changes were added additional modifications

in the base pairing partner within these sequence contexts. This allowed for

understanding more about how effects on base flipping would affect excision by AAG.

DNA Substrates Indicating Base Stacking and Hydrogen Bonding Partners to Hx

DNA substrates were designed to include base stacking changes around the central

damaged base which was base 13 in the 25-mer long substrate. Hx was either flanked by

T -A base pairs (THxA) or G-C base pairs (GHxC), the rationale being that, T-A and G-C

nearest neighbors represent the weakest and strongest base stacking partners respectively

(100). The G-C flanking base pairs may affect Hx accessibility in DNA due to their

intrinsically stronger base stacking. It has been shown previously that hydrogen bonding

within the base pair affected excision of Hx by AAG presumably by increasing or

decreasing the stability of Hx in DNA (75). So, Hx was base paired with T, F or C, within

these sequence contexts. As shown in Figure 5-1, Hx has different hydrogen bonding

interactions with these bases, forming a wobble base pair with T, no base pairs with F,

and a Watson-Crick base pair with C. In contrast, eA, which is also a substrate for AAG,

forms no hydrogen bonding interactions at all (Figure 5-1) and serves as a good control

as seen with the AAG mutants (Chapter 4). The above sequences were incorporated into

25-nucleotides long substrates, with Hx at position 13. The rest of the DNA sequence was

the same as the sequence shown in Table 4-1. Substrates used for experiments were 5'-









end labeled with 32P on the damaged strand and then annealed to the respective

complement oligonucleotides.

The DNA substrate nomenclature is based on the base stacking partners and the

hydrogen bonding partners to Hx or sA throughout this chapter. For example, a duplex

substrate containing Hx with a 5'T and a 3'A in which Hx is paired with T will be

referred to as THxA*T.


F -H
HN- -N-H F
W__/


Hx*T


Hx*F


SA*T


Hx*C


H -


=1


EA*F


H



H-NH
H


EA*C


Figure 5-1. Chemical structures of Hx and EA base paired to thymine, diflorotoluene and
cytosine. Hx forms wobble base pairs with T, no base pairs with F and
Watson-Crick base pairs with C. EA forms no hydrogen bonds with any of the
three base pairing partners.









Base Stacking and Hydrogen Bonding Effects on Hx

The flanking base pairs were designed to create strongest versus weakest base

stacking interactions with Hx in DNA and hence reflect on the ability of the enzyme to

flip EA or Hx. Base excision activity of AAG was measured using a chemical cleavage/

gel assay using labeled, double stranded DNA substrates with T-A or G-C base pairs

flanking Hx, base paired to T. Increasing concentrations of enzyme (ranging from 20nM

to 640nM) were incubated with 5nM substrate at 370C. Aliquots were withdrawn at

several time points from 0 to 80- min., quenched, and analyzed by denaturing PAGE for

product formation. For each enzyme concentration, the reaction course was fit to an

exponential rise to obtain observed rates (kobs) which were used to compare excision

efficiencies of Hx in the two sequence contexts. Increasing concentrations of enzyme

were used until no change in the progress of the time courses were observed, indicating

that single turnover conditions were achieved. For excision of Hx*T with G-C stacking

partners, almost 4 times more enzyme was required to achieve single turnover conditions

compared to Hx*T with T-A stacking partners (Figure 5-2) and the observed rates at these

saturating enzyme concentrations showed that excision of Hx*T was 1.6- fold faster with

T-A partners than with G-C base stacking partners. Rates are summarized in Table 5.1, at

the end of the results section.









5 5









I 20nM hAAG I--80nM hAAG
< 1 : 4 I16M
y







1LI ,
JS m 20nM hAAG BO --0nMhAAG
A 1 60nM hAAG
I* InM-- 541OnM hAAG

10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70
Timefmini TimelmTin

Figure 5-2. Single turnover excision of Hx opposite T with T-A and G-C base stacking
partners. With T-A base stacking partners (left), saturation was observed with
80 and 160nM AAG, whereas with G-C base stacking partners (right), 640nM
AAG was required to approach saturated excision of 5nM HxoT. Saturation
required almost 4- times more enzyme with the stronger base stacking
partners (See text).


Effects of Hydrogen Bonding Partners on Hx Excision in the Strong and Weak Base
Stacking Context

It is possible that making Hx less constrained in DNA may relieve the effects of the

base stacking partners. This can be tested by replacing T with diflorotoluene (F), which

does not form hydrogen bonds with Hx. In previous studies, removing hydrogen bonding

to Hx was seen to improve its excision by AAG (75). In single turnover excision

reactions, measured for 5nM Hx*F with both T-A and G-C base stacking partners with

increasing concentrations of enzyme (20-160nM), it was seen that saturation was

observed at relatively low enzyme concentrations (Figure 5-3). At these saturating

concentrations, when compared to the rates of excision of the same sequences with Hx*T,

Hx*F excision was enhanced. The enhancement was 2-fold for Hx*F over Hx*T with T-A

stacking partners but the enhancement was dramatic for Hx*F over Hx*T with G-C









stacking partners, making Hx*F with G-C partners a better substrate than HxoF with T-A

partners. The excision time course for Hx*F with G-C partners was so fast that the early

time points were not experimentally measurable by hand mixing. It is presumed that the

enhancement in excision rates is at least 10-fold over Hx*T, as assumed from lower

enzyme concentration excision assays.

In cells during replication, polymerases have a propensity to incorporate C,

opposite Hx, with which it can form Watson-Crick hydrogen bonds, much like a G-C

base pair (101-104). This preference makes Hx mutagenic because with another round of

replication, Hx*C potentially becomes an AT--GC transition. Given the observations

with the effect of base pairing partners on Hx excision, it is important to observe what

effects base pairing with C will have on Hx excision, because even when Watson- Crick

base paired with C, Hx is excisable by AAG. In previous experiments, it was shown that

Hx excision was slower opposite C than opposite T (76). To determine how base stacking

partners can affect Hx excision opposite C, single turnover excision reactions were done

with 5nM Hx*C with T-A and G-C base stacking partners using 160 and 320nM AAG.

Under these conditions, kinetics of excision of Hx was very slow (Figure 5-4). Notably,

when base paired to C, Hx excision was unaffected by the base stacking partners, unlike

when opposite T or F. Excision of Hx*C in both sequence contexts essentially proceeded

at the same rate (Table 5-1).










5






S3-

CL
0 2


1 --20nM AAG
c- 1-_ i c1 A A_.
| I-i-OnM AAG
--,,-- 80nM AAG
---160nM AAG

0 1 2 3 4 5 6 7 a
Time (min)






I 3-







S-2nM AAG
M ( w t 40nM AAG
80n M AAG
--16fiOnM AAG

0 0.5 1 1.5 2 2.5 3 3.5 4
Time (min)
Figure 5-3. Single turnover excision ofHx with T-A and G-C base stacking partners
opposite non- hydrogen bonding base pairing partner, F. Excision of Hx*F
was much faster than opposite T. Increasing AAG concentrations of 20nM
(blue squares), 40nM (yellow triangles), 80nM (green squares) and 160nM
(pink circles) were used. With TA base stacking partners, 5nM Hx*F was
excised to saturation by only 40nM AAG (top, yellow triangles). But with
5nm HxeF with G-C base stacking partners, excision was fastest, with
saturation achieved by only 20nm AAG (bottom, blue squares). A shorter
time scale has been shown in these graphs when compared to Figure 5.2, to
emphasize the faster excision achieved for these substrates.














4


3


C-







10 20 30 40 50 60 70 80
Time(min)


Figure 5-4. Single turnover excision of Hx with T-A and G-C base stacking partners
opposite Watson-Crick hydrogen bonding partner, C. Excision of 5nM Hx*C
with T-A base stacking partners did not saturate with 160nM (blue squares) or
320nM (red triangles) AAG. The same trend was seen with G-C stacking
partners with 160nM (green squares) and 320nM (pink circles) AAG. Time
courses for Hx*C excision in both sequence contexts closely mirror each
other, indicating that excision of Hx was not affected by base stacking
partners when Watson-Crick base paired with C.

Effects of Base Stacking Partners on Binding to Hx Substrates by AAG

It is possible that G-C base stacking partners made flipping Hx less favorable than

T-A base stacking partners, by increasing its stability in DNA. The higher molecular

weight band observed in our EMSA is indicative of a flipped base- enzyme complex,

which would give a lower intensity shifted band with G-C stacking partners than with T-

A stacking partners. Lesser intensity shifted bands were seen with the AAG mutants

incapable or less capable of flipping the damaged base (75). To test this conclusion, 5nM

32P- labeled substrates, identical to the ones used for excision assays, were incubated with









0-80nM enzyme and an EMSA was performed to separate free DNA from the higher

molecular weight enzyme- DNA complex, indicative of specific binding. A catalytically

inactive mutant of AAG, E125Q was used in binding assays, to overcome the problem of

loss of substrate due to excision by AAG during the time course of the EMSA. The

binding affinity of the enzyme for Hx*T with T-A base stacking partners was higher than

with G-C base stacking partners. With T-A base stacking partners, at 80nM E125Q,

almost all the substrate around 90%, was bound by the enzyme (Figure 5-5A, upper

panel) whereas, with G-C base stacking partners, binding was less efficient and even at

80nM enzyme, only about 50% of the substrate was bound (Figure 5-5A, lower panel).

This is also consistent with the observation that more enzyme was required to saturate

GHxC*T in excision assays.

Effects of Hydrogen Bonding Partners on Binding to Hx in the Strong and Weak
Base Stacking Context

The same enhancement shown with excision of Hx*F was also seen in improved

binding affinity of E125Q to both substrates in assays performed with 5nM substrate and

0-80nM E125Q. The enzyme-DNA complex band appeared at lower E125Q

concentrations than when opposite T with T-A stacking partners (Figure 5-5B, upper

panel). With G-C base stacking partners, almost all the substrate around 90% was bound

by 40nM enzyme with no free substrate detectable with 80nM enzyme (Figure 5-5B,

lower panel). This is much improved binding when compared to Hx*T in the same

sequence context, in which only 50% of the substrate was bound by 40nM enzyme

(Figure 5-5B, upper panel. The improved binding affinity was more pronounced for the

G-C substrates than for the T-A substrates, as was observed with excision efficiency.








Binding affinity of the enzyme for Hx*C mirrored the excision efficiency. Duplex
substrate (5nM) was incubated with 0-320nM E125Q and then resolved by EMSA. But
binding affinity was very weak for both DNA substrates (Figure 5-5C, upper and lower
panels). A specific enzyme-DNA complex band was hardly visible even with 320nM
E125Q.


THxA*T


THxA*F


THxA*C


Enzyme-DNA
complex --


* M


- -wswMM


Free DNA--

[Enzyme] nM


,.. .


r4 r4TO


i-lin4 0 M
000 00
o i C g( ao


GHxC*T


Enzyme-DNA
complex -b


Free DNA-*

[Enzyme] nM


GHxC*F


APO O O O c0hNTO O
0 rN r4 C C4 ; 00 Cr4


GHxC*C


0 0 00ao
0 '.. C,4 o Ne


Figure 5-5. Electrophoretic mobility shift assays to measure binding of AAG to DNA
containing Hx in different sequence contexts. Binding of E125Q to 5nM
duplex DNA was observed as the appearance of the higher molecular weight
band in EMSAs. Binding to Hx opposite when flanked by T-A stacking
partners (upper panel) and G-C stacking partners (lower panel), with T (A), F
(B) or C (C) base pairing partners are shown. (See text)









Base Stacking and Hydrogen Bonding Effects on EA

Effects of Base Stacking and Hydrogen Bonding Partners on EA Excision by AAG

In previous experiments it was seen that substitution ofF for T opposite sA did not

affect EA excision rates. We proposed that this was due to EA not making hydrogen

bonding interactions with either T or F. Given the large magnitude of enhancement in

excision seen for Hx*F with G-C base stacking partners, it would be interesting to

determine whether base stacking partners may alter the effect ofF on sA excision. Single

turnover excision of sA*T and EA*F, both with G-C base stacking partners were

measured for 5nM labeled substrate and 20nM-320nM AAG. There were few differences

in the time courses, and rates of excision of sA was largely unaffected by substitution of

F for T (Figure 5-6). The rate enhancement was 1.2-fold of EA*F over sA*T, which is

similar to the rate enhancement observed for EA*F over sA*T when flanked by T-G base

pairs (75). Excision of sA was much slower than excision of Hx, irrespective of the

sequence context, highlighting the fact that AAG had a diverse substrate range which

may differ intrinsically in their interaction with the enzyme. EA excision opposite C was

also similar to excision when opposite T and F with G-C stacking partners (Figure 5-7).

In all three sequences sA excision kinetics followed the same time course and showed

similar rates of excision (Table 5-1). Strikingly, excision of Hx opposite C with both T-A

and G-C stacking partners resembled the kinetics of excision of sA in any sequence

context.










5


S4


a3

S2





0
a-2
,


Oa


Time(min)


Time(min)


Figure 5-6. Single turnover excision of sA with G-C stacking partners. 5nM EA*T (left)
and EA*F (right) were used in excision assays with 20 to 160nM AAG to
achieve single turnover conditions. These sequences were used as controls
because sA excision is not affected by base pairing partners. The dramatic
effects seen for Hx excision with G-C stacking partners was not observed for
EA excision, the time courses for both EA*T and EA*F substrates when with
G-C stacking partners were very similar.


5 I '


4


3





-- 160nMAAG
0
I-2


0 ..



10 20 30 40 50 60 70 80
Time(min)


Figure 5-7. Single turnover excision of sA opposite C with G-C base stacking partners.
5nM EA*C was excised by 80 and 160nM AAG. The time courses and rates
were similar to those observed for EA*T and EA*F.










Effects of Base Stacking and Hydrogen Bonding Partners on Binding to EA
Substrates by AAG

No effect was seen on binding affinities to EA*T and EA*F flanked by G-C base

pairs, in EMSAs (Figure 5-8A and B), requiring only 10-20nM enzyme to bind all of the

substrate in both cases. Binding to EA*C DNA was as efficient as binding to the other two

sA substrates (Figure 5-8C).

GcAC*T GsAC*F GeAC*C

Enzyme-DNA
complex



Free DNA WW 1 1 .., W
[Enzyme]nM co coo
WI V-4 elwS V W 0 M in V-4 M V 00 M in V-4 N IV g



Figure 5-8. Binding of E125Q to EA substrates with G-C stacking partners. Binding of 0-
80nM E125Q to 5nM EA*T (A), EA*F (B) and EA*C (C) was observed in
EMSAs. Binding affinity to all three sA substrates was essentially the same,
with almost all DNA bound by 10nM E125Q.



Melting Temperatures of Hx and EA Substrates

The effects of base stacking and hydrogen bonding partners on the excision of Hx

could be predominantly due to altered stability of Hx in the DNA in these sequence

contexts, which in turn would reflect on how efficiently AAG can bind and flip Hx.

These effects were seen in the amount of enzyme required to bring about saturated

excision and the binding to DNA in different sequence contexts. To determine if base









stacking and hydrogen bonding partners bring about altered stability in DNA, melting

temperatures were measured by monitoring the change in UV absorbance at 260nm over

a controlled temperature ramp of 0.5C per minute. A melting curve was obtained whose

first derivative was taken to give a peak corresponding to the Tm. Mean Tm with standard

deviation from two independent experiments for each substrate are given in Table 5-1. As

a general trend, it was seen that the Tm for substrates with Hx and EA opposite C were the

highest when compared to the same bases opposite T or F, within the same flanking base

pairs. For Hx with G-C base stacking partners, the Tm was highest when base paired with

C, intermediate when base paired with T and lowest when base paired with F. Generally,

all three melting temperatures were higher than the corresponding ones for Hx with T-A

base stacking partners. So it can be concluded that, the strong base stacking partners, may

affect the stability of Hx considerably and hence the added effects of hydrogen bonding

or removal of hydrogen bonding are more evident in this sequence context. This also

agrees well with the excision rates of Hx in these sequence contexts. When placed

between T-A base stacking neighbors, differences in Tm are not as distinct as for G-C

neighbors. But the differences in Hx excision rates were also modest in this sequence

context when T was replaced by F. sA did not show any differences in excision when T

was replaced by F, and Tm for the sA substrates are not very varied. It must also be noted

here that EA excision is much slower than Hx excision and may be mechanistically very

different from Hx excision. Hence it is hard to correlate sA excision with the observed Tm

and compare them with the other Tm for Hx substrates. It was also seen that for Hx with

T-A base stacking partners and the sA substrates, the melting temperature was slightly

higher when T was replaced by F. This may be because; F is a good stacker in DNA (105,









106). This stacking property ofF may not affect the flipping effects due to lack of

hydrogen bonds, but may just affect the Tm and hence the observation that for F in these

two sequence contexts, reduced Tm may not necessarily implicate increased excision

rates.

Table 5.1. Melting temperatures of Hx and EA substrates and corresponding single
turnover excision rates
Substrate Base pairing Tm (C) kobs (minl-)*
partner
THxA T 45.50.0 1.50.3
F 46.51.0 2.60.4

C 48.31.3 0.070.01
GHxC T 51.50.5 0.950.07
F 47.80.3 nd

C 53+0.0 0.060.01
GeAC T 47.00.5 0.040.00
F 48.50.0 0.070.00

C 49.30.3 0.0750.007


* kobs values are based on mean single turnover excision rates for two independent
experiments at saturation, with standard deviations.
kobs values for GHxC opposite F were experimentally too fast to measure by hand. (See
text).

Sequence Context Effects and Implications for AAG Activity

The active flipping mechanism of DNA glycosylases is very important for the

overall efficiency of the base excision repair pathway. In the case of AAG, though

flipping is facilitated by the intercalation of Tyr-162 into the helix, how the damaged

base and its interactions in DNA can contribute to recognition, makes an interesting line

of thought. Though the crystal structures of AAG, bound to a pyrrolidine abasic site

analog (77) and 1, N6- ethenoadenine (78) show no specific contacts between the enzyme









with the base opposite the damaged base, excision efficiency of certain damaged bases is

more an identity of the base pair than the base itself (76). It has been shown that AAG

substrates vary in their sensitivity towards the base pairing partner and mutations to Tyr-

162, which may decrease flipping efficiency (75). Only for Hx, removing hydrogen

bonding could rescue the Y162F mutation, to the level of activity of the wild type

enzyme. No rescue occurred for EA. This is a reminder of the diverse substrate range of

AAG and that different steps in recognition and excision contribute to different levels for

the substrates. The thymine analog, difluorotoluene, indicated that increasing the

flexibility of Hx in DNA will also increase its flippability by AAG.

Added to the effect of the base pairing partner, flippability can potentially be

affected by other changes around the damaged base, such as base stacking partners. Base

stacking partners may increase or decrease the stability of base in DNA. In this chapter,

the 5' and 3' base stacking partners to Hx were chosen as possible candidates to change

and thereby affect flippability of Hx by AAG. Either T-A base pairs or G-C base pairs

were chosen as the base stacking partners to represent relatively weak and strong base

stacking neighbors to Hx. A working model for the activity of AAG has already been

discussed based on the results from Chapter 4, in which the enzyme can use a two- step

process to specifically identify and excise its substrates efficiently. In this model, the

enzyme uses the flipping step as an initial test for destabilized bases in DNA, which

could indicate possible substrates. After the base is flipped, AAG uses the factors in the

active site like proper fit in the active site, alignment of the glycosylic bond for cleavage

and suitability of the leaving group that lead to chemistry to finally identify the flipped

base as a substrate and excise it from DNA. According to this model, flipping can be an









important determinant of the enzyme's efficiency. Factors that affect the "flippability" of

a base may in turn contribute to excision efficiency too.

It required about 4 times more enzyme to saturate the rate of excision of Hx*T with

G-C than T-A stacking partners and excision was also 1.6-fold slower than for TA

neighbors. This shows that the strong base stacking contributed by the G-C base pairs

probably increased the stability of Hx*T and made it much more difficult to be flipped by

the enzyme, as can also be seen in the reduced binding affinity. If this were the case, then

removing the constraint of hydrogen bonding must be able to destabilize Hx more and

hence make it more easily flipped. This is exactly what happened when T was replaced

by F, to which Hx cannot hydrogen bond. Both binding affinity and excision efficiency

increase for Hx*F, in both sequences. Most striking was the enhancement of excision

rates seen with G-C stacking partners, which was a 9-fold enhancement, compared to a 2-

fold enhancement with T-A stacking partners. The stronger base stacking partners, G-C

base pairs, seem to have exaggerated the effect ofF on Hx excision by AAG, possibly

owing to the fact that the effect of destabilization of Hx*F on flipping was more

pronounced when in a stronger base stacking sequence. This could mean that the

interactions between AAG and the DNA during the formation of a stable- flipped Hx

complex were stabilized by the strong base stacking partners while at the same time, F

destabilized Hx to enable easier flipping by AAG. The weaker base stacking sequence, T-

A partners, may not have the same effect on the enzyme's interaction with DNA, and

hence the base pairing partner effects on flipping were modest. The assumption that DNA

stability is affected by the base stacking partners is backed by the melting temperatures

measured for the various substrates.









In contrast, EA excision was not greatly altered when placed between G-C base

pairs. The rate of sA excision and the binding of AAG to EA substrates when placed

between G-C stacking partners mirrored the rates and binding properties of sA placed

between T-G partners (75). It has been shown before that EA excision was not affected by

the base pairing partner. Binding was unaltered too. Since sA lacks the ability to

hydrogen bond with any base, the base pairing partners did not add to its destabilization

in any way. It is also possible that its intrinsic instability in DNA makes sA adopt an

extra helical conformation much more easily than Hx; hence making it more accessible to

AAG.

For Hx the wobble base pair with T adds to its stability and impedes flipping,

which can be overcome by other changes that decrease stability. This was seen when Hx

is placed between T-A base pairs and when placed opposite a non-hydrogen bonding

partner. Strikingly though, this neighboring base pair sensitivity was lost when Hx was

Watson-Crick base paired with C. In the cell, Hx is formed opposite T due to

deamination of A but; replicative polymerases have a high propensity to place a C

opposite Hx, which leads to transition mutations. The fact that, when opposite C, Hx

repair is impaired to the same degree irrespective of the base stacking partners indicates

an important biological function. Hx repair opposite C would be mutagenic and a

Watson-Crick base pair may overcome the base stacking partner effect to prevent repair.

So stability of Hx in DNA may be an important factor in deciding its fate, a wobble base

pair may make it more discernible by AAG while a Watson- Crick base pair may make it

less discernible, possibly by shifting the flipping equilibrium. This difference in stability

was partly reflected in the melting temperatures for the various substrates, especially for









the Hx base pairs between GC base pairs, with Hx*C substrates having the highest Tms

and Hx*F substrates having the lowest Tms, with Hx*T being in between. Only a modest

difference in TmS was noticed for the Hx base pairs with T-A stacking partners, adding

weight to the argument that base stacking partners can affect the stability of the Hx base

pair in the substrates.

An important biological outcome of this scenario, in which local sequence context

is seen to affect Hx removal by AAG, is its significance in understanding the presence of

mutational "hot-spots and cold-spots" in the genome. The effect of local sequence

context in the stability of Hx in DNA can play a major role in both enhancing and

reducing its repair. This in turn may contribute to the complex interplay between factors

dedicated to protecting the cell from mutations, like repair mechanisms and the

limitations facing them.














CHAPTER 6
ACTIVITY AND STABILITY OF AAG DURING ASSAYS

The kinetics of AAG revealed a puzzling dilemma. The enzyme was not able to

catalyze multiple rounds of excision. Under multiple turnover conditions, AAG

performed anywhere from one to three turnovers and then seemed to stop catalyzing base

excision. Under multiple turnover conditions, there is excess substrate over enzyme and

ideally, an enzyme goes on catalyzing until all the substrate is depleted. But under these

conditions, factors such as dissociation from product and reassociation with substrate will

affect the catalysis by the enzyme. So single turnover conditions in which product

dissociation will not contribute to the observed rate of catalysis were used in all

experiments to study the properties and mechanism of action of AAG. Under single

turnover conditions, there is a vast excess of enzyme over substrate and ideally,

saturation of substrate is observed. The observed excision rates are a measure of the rate

of excision when substrate is saturated with enzyme and reflects the rate of some step

after binding the substrate and prior to product dissociation. Although single turnover

excision kinetics offers a good measure of AAG's activity, the fact that the enzyme

cannot catalyze several rounds of excision prompted several questions relating to its

activity and stability. Is the enzyme losing activity during the experiment? Is this property

also a function of the substrate and its sequence context? Are reaction conditions not

ideal for AAG? These were important questions to address in order to design experiments

in the future and make the most of the knowledge gained from previous experiments. In

addition, evidence was mounting about similar activities observed for other glycosylases









like human thymine DNA glycosylase (107) and the human MutY homolog (108). These

questions, when answered may reveal some missing links between glycosylase activity

and probably, the BER pathway and its components. So, a systematic experimental

design to address these questions was performed. In these experiments, enzyme death, the

turnover issue as an effect of sequences and other conditions that could improve AAG

activity were considered as possible candidates to test.

Loss of AAG Activity Can Contribute to Reduced Catalysis

AAG loses activity when stored below a final concentration of 40[tM for periods

longer than 6-8 months at -800C. The enzyme also lost activity, both binding and

excision, when stored for more than 10 days at -200C. This indicated that, either the

enzyme had to be stored above a critical concentration to preserve activity or the enzyme

slowly loses activity over time, whatever concentration it may be. This loss of activity

might be more significant at the higher temperature in which excision assays were

performed. To determine if this is true, two simple enzyme "death assays" were

performed. In both assays, 5nM THxA*T was used as the substrate with 40nM AAG.

This was the substrate of choice because Hx was found to be efficiently excised by AAG

but excision was not so fast that hand mixing experiments could not be used (Chapter 5).

For single turnover excision assays, a 4X concentration of the enzyme was needed for the

assay stored in ice and diluted directly into the assay mix to start the reaction. In the first

enzyme death assay, 160nM AAG was pre-incubated for times ranging from 0 to 120

minutes at 370C. Excision assays were performed to determine the loss of activity during

the pre-incubation. Time points were taken as for a normal excision assay and the

quenched samples were analyzed by PAGE and the product formed plotted over time










(Figure 6-1). The products formed and the rate of product formation was compared for

various periods of pre-incubation at 370C (Table 6-1). It was determined that the loss of

activity was not drastic. When compared to the "No pre-incubation" control, there was a

slight drop in the amount of product formed, progressively, with increasing times of

incubation, from 4.8nM to 4.4nM. However the rates of excision did drop with increasing

pre-incubation times, to almost two times lower than the control rate (Table 6-1). Even

after 2 hours at 37C, the loss of activity was much less than the activity of AAG on the

slow substrates, for example, HxeC for which, 160- 320nM AAG was required to

observe comparable activity.


5



S4


0 3




Ca

< 2


a,


I i ''


I-


-- No pre-Incubatlon
---30 min. pre-Incubatlon
---60 min. pre-incubation
-e-120 mln. pre-Incubation
-t--240 mln. pre-Incubation
15 20 25 30 35 40
Time(min)


Figure 6-1. AAG death assay under single turnover conditions. 5nM THxAeT was
excised by 40nM AAG diluted from a 160nm AAG stock which was
incubated at 370C for the indicated times (0 to 240 minutes). Reactions were
started by diluting the stock 4-times into the tube. A serious loss of AAG
activity was not seen even after 240 minutes at 370C.


I,' '' I I, I I











Table 6.1. Comparison of pre-incubation of 160nM AAG at 370C with product formed
and rates of product formation with 5nM THxA*T
Pre-incubation at 370C (min) Product formed (nM) kobs (min1)
0 4.8 0.4
30 4.5 0.3
60 4.5 0.3
120 4.4 0.25
240 4.4 0.2


As there was not a significant loss of enzyme activity upon incubation of the 4X

stock at 370C, another approach was chosen, in which AAG was pre-incubated at 370C

with the assay buffer for the same time periods used before, but at a final assay

concentration of 40nM instead of 160nM. The excision assay was started by adding 5nM

substrate to the pre-incubated AAG. This will also mimic the condition of the usual assay

carried out and may tell us more about whether AAG was losing activity during the assay

itself. So, AAG was pre-incubated at 370C for times ranging from 0-240 minutes and

then the reactions were started by adding DNA to each mix, to give a final enzyme

concentration of 40nM and substrate concentration of 5nM. The experiment was

performed as before and product formed (Figure 6-2) with the single turnover excision

rates (Table 6-2) was obtained. In this case, the observed loss of activity was

considerable. Both the amount of product formed as well as the rate of product formation

was reduced when compared to the "No pre-incubation" control. The progressive

reduction was more pronounced for 40nM AAG than for 160nM AAG. In 60 minutes at

37C, both the amount of product and the rate were only one-third of the "No pre-

incubation" control. After 240 minutes of pre-incubation, there was no detectable product









formed. This means that the enzyme was totally inactive after this extended period at

37C. However a significant drop occurred with every pre-incubation time at 370C

beginning at 30 minutes (Table 6-2), suggesting that AAG could be progressively losing

activity during the time course of our single turnover assays. This may either be due to

enzyme instability or the absence of an additional factor that could stabilize it. This factor

may range from the right pH or ion to another specific protein.

These death assays were an important indication of the limitations faced in our

experiments, the enzyme itself. The additional concentration-dependent loss of activity

highlighted how enzyme death may be more evident when less AAG was used.

Consequently, there could be a critical concentration below which enzyme death could be

rapid. In the traditional multiple turnover assay with an excess of substrate over enzyme,

this phenomenon may be contributing significantly to the lack of turnovers. According to

Selwyn (115), the test for enzyme inactivation during the course of an assay comes from

the super-imposability of the progress curves at any concentration of the enzyme. When

the enzyme loses activity over the progress of the assay, the enzyme itself will follow a

first- order time dependence varying with concentration. Hence the product formed over

time may vary with different enzyme concentrations, giving non- super-imposable

progress curves. This property due to enzyme inactivation is called the "Selwyn's test".

The behavior of AAG at the two different concentrations used, along with the progress

curves obtained during single turnover titrations (Chapter 5) indicate similarities with the

behavior under Selwyn' test. This similarity may indicate possible loss of AAG activity

during the assay and needs to be further investigated for designing strategies to explain

the inactivity and to overcome the effects of inactivity.




















4






0
am 2 1 I




.0 1 --- No pre-Incubation
30min pre-incubation
-*-60min pre-Incubation
---120min pre-lncubatlon
5 10 15 20 25 30 35 40

Time(min)


Figure 6-2. AAG death assay under single turnover conditions. Assays were done with
5nM THxA*T and 40nMAAG which were preincubated at 370C for the
indicated times (0 to 240 minutes). Reactions were started by adding substrate
to preincubated AAG. A considerable loss of AAG activity was seen starting
at 30 minutes to no detectable activity after 240 minutes.









Table 6.2. Comparison of pre-incubation of 40nM AAG at 370C with the product formed
and rate of product formation with 5nM THxA*T
Pre-incubation at 370C (min) Product formed (nM) kobs (min-1)
0 4.1 0.3
30 3.8 0.2
60 3.5 0.1
120 2.0 0.1
240 Not detectable Not detectable


Multiple Turnover of Hx Is Dependent on the Base Pairing Partner

Based on the previous results which revealed base pairing and base stacking

sensitivities in AAG mediated excision of Hx, it was clear that Hx excision depends more

on local sequence context than EA. Under multiple turnover conditions, using a THxG

substrate with T being the base pairing partner to Hx, approximately two or three

turnovers were always observed. This meant that, AAG was able to catalyze more than

one enzyme equivalent, but is limited by other factors and then is not able to catalyze

excision of all the substrate. Removing hydrogen bonding to Hx was seen to improve its

excision, especially with strong base stacking partners. A multiple turnover excision

assay, in which 50nM Hx with GC stacking partners was used, opposite either T or F

with 5nM AAG was performed. When opposite F, almost 45nM substrate was excised

completing about 9 turnovers for AAG. On the other hand, the same substrate opposite T

showed just 3nM substrate excised, meaning that AAG was unable to complete more than

half a turnover (Figure 6-3). This was even less than the usual number of two or three

turnovers seen for Hx*T substrates with TG base stacking partners. This inhibition of

turnovers for Hx with GC stacking partners opposite T was observed again when 20nM

AAG was used with 50nM substrate, while the same substrate opposite F was always









fully excised. In multiple turnover experiments done with the previous THxG substrate,

which should be intermediate between TA and GC stacking sequences, F as the base

pairing partner stimulated complete excision of Hx. Whereas T as the base pairing partner

gave about two or three turnovers (Figure 6.4).


20 30
Time (min)


Figure 6-3. Multiple turnover of Hx*T and Hx*F with G-C stacking partners. 50nM
substrate and either 5 or 20nM AAG were used in these assays. HxOF was
turned over completely by both concentrations of enzyme, meaning that AAG
was able to do multiple turnovers of Hx when not hydrogen bonded. On the
other hand, excision of Hx*T showed only half a turnover. This restriction on
AAG turnovers seemed to be sensitive to the hydrogen bonding partner.









50



40

c-


S30
.0




-w- Hx-T
Hx-F
10 20 30 40 50 60
Time (min)



Figure 6-4. Multiple turnover of HxoT and Hx*F with TG stacking partners. 50nM
substrate and 10nM AAG were used. In the presence of stacking partners,
intermediate between TA or GC base stacking partners, Hx*T excision
showed around two or three turnovers with AAG, whereas, Hx|F was excised
to completion by AAG.

There seemed to be a clear connection between the stability of Hx in DNA and the

ability of AAG to turnover Hx. This could mean that the extremely fast excision of HxoF

in any sequence context is over shadowing the combined effects of loss of AAG activity

and any other modes of inhibition possible in a multiple turnover reaction. A multiple

turnover reaction may reflect product interactions with the enzyme that are not a factor in

a single turnover reaction. So, the differences in turnover seen for Hx depending on the

sequence context may be an exaggeration of the enzyme being slow due to the strong

base stacking and hydrogen bonding sequence context and at the same time losing

activity rapidly. It must also be recalled that in the stronger base stacking sequence,

binding is much less efficient. The sequence context of the substrate DNA may play an









important role in taking into account product inhibition issues, which may make much

less enzyme available to bind the substrate in addition to reduced binding affinity.

Multiple and Single Turnover of EA Present Different Pictures

Sequence context had little effect on the excision of sA. Whatever may be the base

stacking or base pairing partners to EA, under single turnover conditions, product formed

and the rate of product formation were virtually unchanged. This same behavior was

observed under multiple turnover conditions too. Whatever the sequence context, exactly

one equivalent of the amount of AAG used was excised when sA was the substrate.

Whether it was sA opposite T or F, only one turnover was seen using GeAC DNA. 50nM

substrate and 5 or 10nM AAG were used in multiple turnover reactions as described

above. It was surprising that compared to the progress of single- turnover reactions, the

burst of product to reach one turnover was much faster under multiple turnover

conditions. Therefore, a titration of AAG to span multiple turnover and single turnover

conditions was done with 5nM EA*T with GC stacking partners, the same substrate used

before. It was seen that the lower the enzyme concentration, the faster the burst of

product, though under multiple turnover conditions, only one turnover was seen (Figure

6-5). As the AAG concentration reached single turnover levels, above 5nM, the product

formation curve resembled previous single turnover progresses and the rates of single

turnover were much lower than multiple turnover. It was also observed that, as AAG

concentration increased in the multiple turnover part of the titration, rates dropped

whereas, in the single turnover part of the titration, rates remained constant throughout

the concentration range (Table 6-3).









The above observations appeared to conflict with those made previously by Dr.

Clint Abner, who conducted a similar titration with 50nM EA DNA and 10- 800nM

AAG, final concentrations. He observed a concentration dependent rise in product

formed consistent with a saturation curve. Interestingly, the observed rates of excision

showed some anomalies with his experiments too, with rates dropping for the 50 and

100nM AAG experiments and remaining more or less constant for the other single

turnover experiments. Together our results may indicate an AAG-dependent change in

the excision kinetics of sA which needs further investigation to explain.


30 40 50 60 70 80
Time(min)


Figure 6-5. Multiple to single turnover titration of AAG with EA*T. 5nM substrate was
used with 1.25 and 2.5nM AAG (multiple turnover conditions), 5nM AAG
(equal substrate and enzyme) and 10, 20 and 40nM AAG (single turnover
conditions). One turnover was seen at a faster rate under multiple turnover
conditions (See text and Table 6.3)











Table 6-3. Observed rates of multiple to single turnover titration assays of AAG with
5nM EA*T
AAG (nM) 1.25 2.5 5.00 10.0 20.0 40.0
kobs (min-1) 0.2 0.2 0.08 0.1 0.1 0.1
Product (nM) 1.0 1.8 2.60 0.4 0.6 1.2

Optimization of Assay Conditions for Maximum AAG Activity

The loss of AAG activity at 370C may be due to non- optimum assay conditions,

which may destabilize AAG especially at higher temperatures. Variations in buffer

composition including pH, salts and other stabilizing agents like BSA may improve

stability. A recent publication has suggested that for neutral substrates like Hx and EA,

pH optimum for efficient excision by AAG is 6.0. The pH optimum is 6.0 for neutral

substrates to make them favorable leaving groups upon excision (109). The assay buffer

that we routinely use was based on previous data and stability assays and was at a pH of

8.0. To determine if excision was more efficient at pH 6.0, assays were done at pH 6.0,

with sodium acetate pH 6.0/sodium chloride or potassium acetate pH 6.0/potassium

chloride instead of HEPES pH 8.0/potassium chloride, which were the buffer and salt

used in all our assays. Assays contained 50mM buffer and 100mM salt. The potassium

buffer and salt were used at pH 6.0 because it was shown previously that potassium

chloride was more suitable for storing and assaying AAG than sodium chloride. KC1

stabilized AAG better than NaC1. An excision reaction was performed under these

conditions with 5nM Hx*T with G-C base stacking partners and 50mM AAG diluted in

the respective storage buffer to maintain salt and pH conditions. The substrate was so

chosen that any increase or decrease in activity will be very evident, this being the









slowest excised substrate of all and probably the most stable substrate. The observations

were interesting though by no means reflective of AAG activity being improved at pH

6.0. Only the potassium acetate pH6.0/potassium chloride buffer showed more products

formed. The sodium acetate pH 6.0/sodium chloride buffer showed activity similar to the

HEPES pH 8.0/potassium chloride, which was the previously used buffer/salt

combination (Figure 6-6). The rate of product formation though remained essentially

unchanged under all three conditions (Table 6-4). The argument that the right pH was not

achieved to ensure maximum activity of AAG was not supported by these experiments.

The effect of the potassium salts may be due to some other effect of salt on DNA

structure and stability. To verify this, melting temperatures were measured as described

for the same substrate used in these assays in the respective buffers. The overwhelming

observation was the lower Tm with Potassium acetate, pH 6.0/Potassium chloride. The Tm

was lower by about 30C than the Tm of 51.50C seen with HEPES, pH8.0/Potassium

chloride and Sodium acetate, pH 6.0/Sodium chloride buffers (Table 6-4). Therefore, the

effects of the potassium buffer may not be a pH effect but a salt effect on DNA stability

or structure which may make Hx more flippable by AAG. The buffers used by the

authors (113, 114) to illustrate the effects of lower pH on neutral substrates did not hold

in the case of our assays since no enhanced excision of Hx was observed in the sodium

buffer at pH 6.0 over our buffer at pH 8.0.