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Interaction of Adenomatous Polyposis Coli (APC) and Base Excision Repair Proteins

Permanent Link: http://ufdc.ufl.edu/UFE0022605/00001

Material Information

Title: Interaction of Adenomatous Polyposis Coli (APC) and Base Excision Repair Proteins Its Implication in Colorectal Carcinogenesis
Physical Description: 1 online resource (107 p.)
Language: english
Creator: Armas, Melissa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: adenomatous, apc, beta, cancer, coli, colon, colorectal, dna, endonuclease, fen, flap, polymerase, polyposis, repair
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mutations in the adenomatous polyposis coli (APC) gene are among the earliest events which lead to the development of colorectal cancer. The APC protein is critical for the maintenance of the dynamics of colonic epithelial cells. APC plays a role in the regulation of beta-catenin, cell cycle control, cell-cell adhesion, chromosomal stability, chromosomal segregation, and apoptosis. Previous studies have shown that the APC gene is upregulated in response to DNA damage. However, the biological significance of the DNA damage induced APC levels is not yet known. We hypothesize that the DNA damage-induced APC levels may play a role in DNA repair and hence may be involved in the early stages of initiation of colorectal carcinogenesis. Interestingly, it has recently been found that APC can interact with two base excision repair (BER) proteins, DNA polymerase beta? (pol-beta) and Flap endonuclease 1 (Fen-1), and block pol-beta-directed BER. In the present study, the critical amino acid residues of pol-beta and Fen-1 required for their interaction with APC have been identified. Furthermore, two mechanisms for blockage of long patch (LP)-BER by APC through its interaction with Fen-1 have been discovered.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Melissa Armas.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Narayan, Satya.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022605:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022605/00001

Material Information

Title: Interaction of Adenomatous Polyposis Coli (APC) and Base Excision Repair Proteins Its Implication in Colorectal Carcinogenesis
Physical Description: 1 online resource (107 p.)
Language: english
Creator: Armas, Melissa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: adenomatous, apc, beta, cancer, coli, colon, colorectal, dna, endonuclease, fen, flap, polymerase, polyposis, repair
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mutations in the adenomatous polyposis coli (APC) gene are among the earliest events which lead to the development of colorectal cancer. The APC protein is critical for the maintenance of the dynamics of colonic epithelial cells. APC plays a role in the regulation of beta-catenin, cell cycle control, cell-cell adhesion, chromosomal stability, chromosomal segregation, and apoptosis. Previous studies have shown that the APC gene is upregulated in response to DNA damage. However, the biological significance of the DNA damage induced APC levels is not yet known. We hypothesize that the DNA damage-induced APC levels may play a role in DNA repair and hence may be involved in the early stages of initiation of colorectal carcinogenesis. Interestingly, it has recently been found that APC can interact with two base excision repair (BER) proteins, DNA polymerase beta? (pol-beta) and Flap endonuclease 1 (Fen-1), and block pol-beta-directed BER. In the present study, the critical amino acid residues of pol-beta and Fen-1 required for their interaction with APC have been identified. Furthermore, two mechanisms for blockage of long patch (LP)-BER by APC through its interaction with Fen-1 have been discovered.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Melissa Armas.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Narayan, Satya.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022605:00001


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THE INTERACTION OF ADENOMATOUS POLYPOSIS COLI (APC) AND BASE
EXCISION REPAIR PROTEINS: ITS IMPLICATIONS IN COLORECTAL
CARCINOGENESIS




















By

MELISSA LIZ ARMAS


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

2008






































O 2008 Melissa Liz Armas
































To my parents, Eser and Mayra, for always believing in me; In memory of my Grandmother
Maria, for inspiring me and teaching me to always have faith; In memory of my Grandfather
Victor whose battle with cancer inspired me to pursue my doctorate









ACKNOWLEDGMENTS

I thank God for allowing me to finish this journey and being with me every step of the

way. I thank my parents, Eser and Mayra Armas, for their guidance, love, support, and

encouragement. I thank my family and friends for their unfaltering prayers and encouragement. I

thank my mentor Dr. Satya Narayan, for giving me the opportunity to pursue my doctoral

research in his laboratory and for sharing his knowledge with me. I thank Dr. Aruna Jaiswal for

his experimental guidance and assistance.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF FIGURES .............. ...............8.....


AB S TRAC T ............._. .......... ..............._ 10...


CHAPTER


1 INTRODUCTION ................. ...............11.......... ......


Colorectal Cancer .................. ............... ...............11......
Adenomatous Polyposis Coli (APC) ................. ......... ...............11. ...
The APC gene.................... ........... .. ...............11.....
The APC Protein and its Many Functions ................. ...............12........... ..
APC and p-catenin regulation .........._.... ...............13..._.__. .....
APC and its role in cell adhesion ................. ...............15........... ..
APC and its role in cell migration ................. .. ........ ... ... ......... ..........1
Role of APC in chromosomal segregation and mitotic spindle assembly ........._....17
Role of APC in cell cycle regulation and apoptosis .........._.. .......... ..............18
APC and its involvement in DNA damage and repair ................. ............. .......19
DNA Damage and Repair ............ ..... ._ ...............20...
DNA Damage Checkpoints .............. ...............20....
G1/S checkpoint............... ...............2
S-phase checkpoint............... ...............2
G2/M checkpoint .............. ...............22....
DNA Repair............... .. ...............2
Direct damage reversal ................. ...............23....... ......
Nucleotide excision repair............... ...............23.
Mismatch repair............... ...............23.
Double-strand break repair ......_._................. ........_._ ......... 2
Base excision repair .............. ...............25....
DNA Polymerase P ( pol-P) .............. ...............27....
Pol-P Structure ........_................. ...............28.......
Roles of Pol-P ........._...... .. ...___ .... ... .. ._ ....... ... ..... .......3
Post-translational Modifications of Pol-P and its Effect on Pol-P Function ........._........31
Flap Endonuclease 1 (Fen-1) ................... .. ....... ...............31.....
Fen-1 Structure and Biochemical Characteristics .............. ... .. ...............3
Residues and Stuctural Motifs of Fen-1 Important for Fen-1 Function ................... .......33
Roles of Fen-1 .................. ........... ........... .......... ............... .............33
Post Translational Modifications of Fen-1 ................ ...............35...............
Purpose/Significance .............. ...............36....












2 MATERIALS AND METHODS .............. ...............42....

Yeast Cell Maintenance ................ .. ....___.. ....... ._. .. ....... .. .......4
Yeast Two-Hybrid Assays of the Interaction of APC with Pol-P and Fen-1 .........._.............42
Maintenance of Mammalian Cells ........._...... ...............43..__._. .....
Generation of Pol-P Deletion Constructs .............. ...............43....
Site Directed Mutagenesis of Pol-0.......... ..... ......___ .. .......__ .........__ ....... 4
Yeast Two-Hybrid Assay of Pol-P Deletion Constructs, Pol-P Mutants, and APC ...............44
APC Peptides ............... .. ..... .. ...... .............. .. .............4
Synthesi s and Labeling of the Fen- 1 Sub state. ...._._._.. .... .._.... ...............45.
Fen-1 Activity in vitro ................. ... .... .... . ... ..........4
PCR of Fen-1 Protein Fragments for use in in vitro Transcription/Translation....................46
Site-Directed Mutagenesis of Fen-1 .............. ..... .. ... ....... ................... .............4
Synthesis of Fen-1 Protein Fragments, Fen-1 wt, and Fen-1 Mutant Proteins Using in
vitro Transcription/Translation .............. ...............49....
Far-W western Blot Analysis ............. .. ... .......... ....... ... ........................4
Pull-Down of Fen-1 wild type and FenS-1 K365A/K366A/K367A from TNT Synthesis
Reaction M ixture ........._._ ...... .... ...............50...
Im munohistochem istry .............. ......... ... ...... ...................5
Western Blot Analysis of Fen-1 and APC Protein Levels in the Cytoplasm and the
Nucleus in Cells Treated with DNA-Alkylating Agents .............. ....................5

3 APC INTERACTS WITH DNA POLYMERASE BETA .............. ...............52....


Amino acids Thr79/Lys81/Arg83 of pol-P are Critical for its Interaction with APC .........._.52
Amino Acids Il259A and Y1262A, but not Q1256A are Required for APC's Interaction
with DNA Polymerase P (Pol-P)............... ...............53

4 APC INTERACTS WITH FLAP ENDONUCLEASE 1 (FEN-1) ................. ................ ...59

APC and Fen-1 interact in vivo................... ......... ...............5
The Interaction Site of APC is the Same for Fen-1 and Pol-P ........._. ....... ._. ............59
APC Blocks the Endonuclease and Exonuclease Activities of Fen-1 .............. .... .................60
Amino Acids K3 65/K3 66/K3 67 are Important for the Interaction of Fen-1 with APC.........61
Fen-1(K3 65A/K3 66A/K3 67A) Mutant Retains Catalytic Activity ................... ................... ..63
APC Prevents the Entry of Fen-1 into the Nucleus after Exposure to DNA-Alkylating
A gents .............. ...............64....

5 DI SCUS SSION ................. ...............8_ 3......._ ....


The Interaction of APC and DNA Polymerase P and its Implications in BER ....................83
The Interaction of APC with Fen-1 and its Implications in BER ................. ............... ....84
Conclusions and Future Implications .............. ...............90....














LIST OF REFERENCES ................. ...............92................


BIOGRAPHICAL SKETCH ................ ............. ............ 107...











LIST OF FIGURES


FiMr page

1-1 Roles of APC .......... ................ ...............38. ....

1-2 Structure of the APC protein ................. ...............39...............

1-3 Base excision repair ................. ...............40................

1-4 Structure of Fen-1 .............. ...............41....

3-1 Interaction of APC with pol-P-deletion constructs ....._._._ ..... ... .__ ......._.........55

3-2 Ribbon representation of pol-P highlighting the position of key mutants .......................56

3-3 Interaction of APC with pol-P mutants ...._.. ...._._._.. .......__... ...........5

3-4 Interaction of APC mutants with pol-P. .....___ ..........._.. ........._........._......58

4-1 Interaction of Fen-1 and APC. ............. ...............69.....

4-2 Interaction of APC mutants with Fen-1 ......___ ...... ...............70 ...

4-3 APC blocks the 5'-flap endonuclease activity of Fen-1 ................. ............... ...._...71

4-4 APC blocks the 5'-flap endonuclease activity of Fen-1 regardless of flap size ................72

4-5 APC blocks the 5'-3' exonuclease activity of Fen-1 ......_._.__ .. .... ..... ........._......73

4-6 Protein levels of in vitro transcription/translation of wild-type and fragments of Fen-
1............... ...............74...

4-7 Far-western analysis of the interaction of APCwt and APC(I-A,Y-A) peptides and
Fen-1 protein fragments ................. ...............75................

4-8 Protein levels of in vitro transcription/translation of wild-type and mutant Fen-1............76

4-9 Far-western analysis of the interaction of Fen-1 wild-type and mutant proteins ........._....77

4-10 Fen-1 (K365A/K366A/K367A) mutant retains 5'-flap endonuclease and 5'-3'
exonuclease activity ................. ...............78.......... .....

4-11 Imunohistochemical analysis of APC and Fen-1 co-localization in HCT-1 16-
APC(WT) and HCT-116-APC(KD) cells ................. ...............79...............

4-12 A time-course analysis of co-localization of APC and Fen-1 in colon cancer cells
treated with 1VM S. ............. ...............80.....











4-13 A time-course analysis of co-localization of APC and Fen-1 in LoVo cells treated
with M M S. ............. ...............81.....

4-14 A time-course analysis of nuclear and cytosolic protein levels of APC and Fen-1 in
MMS-treated colon cancer cells .............. ...............82....









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

THE INTERACTION OF APC AND BASE EXCISION REPAIR PROTEINS: ITS
IMPLICATIONS IN COLORECTAL CARCINOGENESIS,

By

Melissa Liz Armas

August 2008

Chair: Satya Narayan
Major: Medical Sciences-Molecular Cell Biology

Mutations in the adenomatous polyposis coli (APC) gene are among the earliest events

which lead to the development of colorectal cancer. The APC protein is critical for the

maintenance of the dynamics of colonic epithelial cells. APC plays a role in the regulation of P-

catenin, cell cycle control, cell-cell adhesion, chromosomal stability, chromosomal segregation,

and apoptosis. Previous studies have shown that the APC gene is upregulated in response to

DNA damage. However, the biological significance of the DNA damage induced APC levels is

not yet known. We hypothesize that the DNA damage-induced APC levels may play a role in

DNA repair and hence may be involved in the early stages of initiation of colorectal

carcinogenesis. Interestingly, it has recently been found that APC can interact with two base

excision repair (BER) proteins, DNA polymerase P (pol-P) and Flap endonuclease 1 (Fen-1), and

block pol-P-directed BER. In the present study, the critical amino acid residues of pol-P and Fen-

1 required for their interaction with APC have been identified. Furthermore, two mechanisms for

blockage of long patch (LP)-BER by APC through its interaction with Fen-1 have been

discovered.









CHAPTER 1
INTTRODUCTION

Colorectal Cancer

Colorectal cancer is the second to fourth most common cancer in the industrialized world.

It is estimated that 148,810 new cases of colorectal cancer will occur in 2008. Of these, 49,960

will result in death (Society, 2008American Cancer Society). Colorectal cancer is usually divided

into two forms: familial or hereditary cases and sporadic cases. Of the hereditary cases there are

two types which are characterized: hereditary non-polyposis colorectal cancer (HNPCC) and

familial adenomatous polyposis (FAP). HNPCC is caused by mutations in genes encoding for

DNA mismatch repair proteins. At least four genes have been shown to cause HNPCC: MSH2,

MLH1, MSH6, and PMS2 (Baglioni and Genuardi, 2004). FAP arises almost exclusively from

germline mutations in the adenomatous polyposis coli (APC) gene. Interestingly, loss of APC

function is an extremely early event in the development of sporadic colorectal tumors. This

suggests that APC is critical in the maintenance of the colon (Nathke, 2004).

Adenomatous Polyposis Coli (APC)

The APC gene encodes a large multi-domain protein which plays integral roles in many

aspects of the cell. Traditionally known as a tumor suppressor, APC plays a role in many

functions that are critical for cell survival. Interestingly, somatic mutations in the APC gene are

found in approximately 80% of all sporadic colorectal tumors. These mutations almost always

result in a truncated APC protein product which lacks some of the critical tumor suppressor

functions of APC such as p-catenin regulation.

The APC gene

The APC gene is located on chromosome 5q21-q22, consists of 8535 nucleotides and

spans 21 exons. The APC gene encodes a 3 10 kDa protein which is composed of 2843 amino









acids in its common isoform (Jaiswal et al., 2004). Approximately 75 percent of the coding

sequence is located on exon 15, which appears to be the most common target for both germline

and somatic mutations ofAPC (Beroud and Soussi, 1996).

Hypermethylation of the APC gene promoter has been observed in colorectal, melanoma,

lung, oesophogeal, gastric, pancreatic, and hepatic cancers (Clement et al., 2004; Esteller et al.,

2000; Grote et al., 2004; Tsuchiya et al., 2000). Methylation occurs at CpG sites in the promoter

region of the APC gene and is mediated by the enzyme DNA methyl transferase 1 (DNMT 1).

DNMT 1 induces methylation of the APC promoter by methylating the CpG region around the

CCAAT-box. Once methylated, chromatin conformational changes occur which block the

binding of the transcription factor CBF to the CCAAT box, silencing APC gene expression

(Deng et al., 2004). Intriguingly, it has been found that DNM~T1 gene expression is regulated by

APC mutations in an indirect manner (Campbell and Szyf, 2003). The above findings indicate

that hypermethylation of the APC promoter may provide an alternative mechanism of APC gene

inactivation during early stages of carcinogenesis.

The APC Protein and its Many Functions

APC is a large multi-domain protein, which is present in both the cytoplasm and the nucleus.

It serves many functions in the cell as demonstrated by its structure (Fig. 1-2). Along the N-

terminus an oligomerization domain, a homology domain, an armadillo repeat region and two

nuclear export signals are present. The oligomerization domain has been shown to be a binding

site for APC mutants. Studies have shown that APC mutant proteins that retain at least the first

171 amino acids are able to bind to this site and produce a dominant negative effect (Dihlmann et

al., 1999; Su et al., 1993). However, a study involving the overexpression of a truncated APC

protein in a mouse model seemed to contradict these findings when the mice did not form tumors

(Smits et al., 1999). The armadillo repeat has been designated a protein-protein interaction site









and has been shown to bind PP2A, Asef, and KAP 3 (Jimbo et al., 2002; Kawasaki et al., 2000;

Seeling et al., 1999). A number of 15-and 20-amino acid repeats are found in the center of the

protein which have been shown to bind p-catenin. Nestled in between the amino acid repeats are

axin binding sites, nuclear export signals, and the mutation cluster region. The mutation cluster

region (MCR) is located between codons 1280-1513 and harbors over 60% of all somatic

mutations in the APC gene (Miyoshi et al., 1992). Mutations that occur in the MCR result in a

truncated protein that lacks most of the C-terminus, which is home to several important sites on

the APC protein. It contains the basic domain, which is the site of microtubule binding, and

binding sites for the proteins EB 1 and hDLG (Bienz, 2002; Erdmann et al., 2000; Matsumine et

al., 1996; Polakis, 1997; Su et al., 1995). Due to its numerous interactions with a variety of

proteins it is expected that APC plays various roles in the cell (Figure 1-1).

APC and j3-catenin regulation

The involvement of APC in the regulation of p-catenin occurs through several routes.

APC is part of the destruction complex, which is activated in the absence of a Wnt signal, along

with axin and glycogen synthase kinase-3-P(GSK3-P). APC causes phosphorylation and

destruction of p-catenin through the ubiquitin proteasome pathway (Peifer and Polakis, 2000). A

recent study suggests that different domains of APC are involved in the regulation of

phosphorylation and ubiquination of p-catenin (Yang et al., 2006). The destruction of p-catenin

keeps it from activating the T cell factor (Tcf) family of transcription factors. Targets of this

family of transcription factors include the oncogene c-myc, cyclin DI, the oncogene c-jun, and

the matrix metalloproteinase matrilysin (Crawford et al., 1999; Nateri et al., 2005; Orford et al.,

1997; Shtutman et al., 1999; Tetsu and McCormick, 1999). When APC is mutated, it prevents

GSK3 P-mediated phosphorylation of p-catenin, sparing p-catenin from ubiquitination and










proteasomal degradation. The stable p-catenin accumulates in the nucleus and binds with the Tcf

family of transcription factors leading to aberrant gene expression, which is associated with cell

proliferation and cell cycle progression (Henderson and Fagotto, 2002). Furthermore, APC has

been shown to be involved in the export of p-catenin from the nucleus, thus reducing its ability

to interact with Tcf transcription factors (Henderson, 2000). APC has also been shown to

sequester p-catenin in the nucleus with the aid of C-terminal binding protein (CtBP), and once

again, prevent its binding to Tcf transcription factors (Hamada and Bienz, 2004). Similarly,

cytoplasmic APC has been shown to sequester cytoplasmic p-catenin. When levels of

cytoplasmic p-catenin are excessively elevated, it induces phophorylation of APC by casein

kinasel, GSK3 P and unknown kinases. Once phosphorylated, APC is able to retain p-catenin in

the cytoplasm by keeping it bound in a complex preventing the entry of p-catenin into the

nucleus and the activation of Tcf signaling (Seo and Jho, 2007). Moreover, APC's role in P-

catenin regulation suggests a role for APC in cell cycle progression. A pathway has been

elucidated which shows that APC can regulate Cdk4 expression as a result of its interaction with

p-catenin. Through the use of an inducible APC gene in HT29 colon cancer cells, He and

colleagues (1998) discovered that c-myc was a target gene of the APC pathway. They found that

when APC was induced c-myc expression was repressed (He et al., 1998), and that c-myc

expression was activated through Tcf-4 which is activated by p-catenin. These results lead to a

proposed pathway for c-myc expression in colorectal epithelial cells. In normal colorectal

epithelial cells, wild-type APC prevents p-catenin from forming a complex with Tcf-4 and

activating c-myc. However, in colorectal tumors which have an APC mutation or activating P-

catenin mutation, P-catenin/Tcf-4 complex formation is increased leading to overexpression of c-

myc and the promotion of neoplastic growth (He et al., 1998).









Further studies proved that Cdk4 is the target of the aforementioned pathway (Hermeking

et al., 2000). After c-nryc overexpression was carried out in human umbilical vein cord cells

(HUVEC) using adenovirus, a transcript encoding Cdk4 was found to be induced. This proved to

be most intriguing since Cdk4 is involved in cell cycle regulation. To further confirm these

results a mutant c-nryc was overexpressed in HUVEC cells to see if the Cdk4 transcript was still

induced. As expected, Cdk4 failed to be induced in the presence of the mutant c-nzyc. These

results suggest that c-nryc is involved in the direct regulation of Cdk4 mRNA. Moreover, a

concordant increase in c-nryc and Cdk4 expression is found in colorectal cancers when compared

to normal colon cells (Hermeking et al., 2000). Based on these data the aforementioned model

with APC has been amended. In normal colorectal epithelial cells, wild-type APC prevents P-

catenin from forming a complex with Tcf-4 and activating c-nzyc. Since c-nryc is not activated,

Cdk4 will not be activated and this prevents cdk4 from phosphorylating and down-regulating

important tumor suppressor genes such as the retinobla~stonza (Rb) gene. However, in colorectal

tumors which have an APC mutation or an activating p-catenin mutation, P-catenin/Tcf-4

complex formation is increased leading to over expression of c-nzyc. Over expression of c-myc

leads to an overexpression of Cdk4, which can phosphorylate the Rb gene causing it to become

down-regulated and promote neoplastic growth.

APC and its role in cell adhesion

APC is a part of the cell-cell adhesion complex with E-cadherin, since it directly binds to

y-catenin and actin filaments as well as p-catenin. In Drosophila mutational inactivation of APC

causes delocalization of p-catenin, gaps between adhesive plasma membranes, and a

mislocalization of oocytes resulting from a failure of cadherin based adhesion (Hamada and

Bienz, 2002). Moreover, Hughes et al., found that enterocytes from C57BL/6J-min/+ (Min/+)










mice showed a reduced association between E-cadherin and p-catenin in adherens junctions

(Hughes et al., 2002). Min/+ mice serve as an animal model for familial adenomatous polyposis.

These mice have one APC allele which carries a truncation mutation at codon 850 and one

normal APC allele (Su et al., 1992). Furthermore, Faux and colleagues were able to show that

restoration of full length APC into SW480 cancer cells, which contain truncated APC, resulted in

enhanced cell adhesion (Faux et al., 2004). The enhanced cell adhesion was attributed to the

involvement of APC in post-translational regulation of E-cadherin localization. When full length

APC was restored E-cadherin localization changed from the cytoplasm to the cell membrane

resulting in tighter adherens junctions (Faux et al., 2004).

APC and its role in cell migration

Wild-type APC is necessary for the upward movement of colonic epithelial cells along the

crypt villus axis. During migration colonic epithelial cells are differentiated into absorptive,

secretary, paneth, and endocrine cells. Mutation or loss of APC causes aberrant migration of

these cells and the formation of polyps as well as altered differentiation in colonic epithelial cells

(Moss et al., 1996; Sansom et al., 2004). Kroboth and colleagues showed that loss of APC results

in immediate defects in cell migration in cells that constitutively- lack APC and in cells where

APC loss was induced conditionally using a Cre-Flox system (Kroboth et al., 2007). Migration

was affected in both cell monolayers and in single cell movements.

Furthermore, APC can regulate cell polarity and migration through the actin cytoskeleton.

APC can interact with a Rac-specific guanine nucleotide exchange factor (Asefl) through its

armadillo repeats. Once activated, Asef activates Rac 1, leading to increased cell spreading,

membrane ruffling and lamellipodia formation (Hanson and Miller, 2005; Kawasaki et al., 2000).

Additionally, it has been shown that truncated APC constitutively activates Asef. The









constitutively active Asef causes a decrease in E-cadherin mediated cell-cell adhesion and

promotes cell migration (Kawasaki et al., 2003). A recent study has shown that APC can also

interact with Asef2, a relative of Asefl. Through this interaction it was found that APC activates

Cdc42, which is involved in actin polymerization and cell polarity (Kay and Hunter, 2001).

Moreover, it has been found that IQ-motif containing GTPase activation protein 1 (IQGAPl), a

scaffold protein involved in the control of actin filaments and microtubules as well as an effector

ofRac 1 and Cdc42, recruits APC to specific sites in migrating cells. A complex is then formed

between IQGAP1, APC, Rac 1, Cdc42, and the microtubule stabilizing protein CLIP-170. If

either APC or IQGAPI1 is removed, polarized migration as well as formation of the actin

meshwork is inhibited (Aoki and Taketo, 2007; Briggs and Sacks, 2003; Watanabe et al., 2004).

Role of APC in chromosomal segregation and mitotic spindle assembly

The ability of APC to interact with Bub kinases and microtubules suggests a role for APC in

chromosomal segregation and mitosis. APC has been found at the ends of mictrotubules in a

complex with the checkpoint proteins Bub 1 and Bub3, suggesting a role of APC in mitosis. It has

been shown that APC localizes to spindle poles, spindle microtubules, and kinetochores

(Dikovskaya et al., 2004; Fodde et al., 2001; Kaplan et al., 2001; Louie et al., 2004) and the loss

of APC or expression of APC truncation mutants, similar to those found in cancer cells, leads to

defects in the mitotic spindle. Moreover, the spindle checkpoint is compromised allowing cells to

progress through mitosis with incomplete chromosomal alignment or attachment (Dikovskaya et

al., 2004; Draviam et al., 2006; Green and Kaplan, 2003; Green et al., 2005; Tighe et al., 2004).

Incomplete chromosomal alignment or attachment can lead to aneuploidy and tetraploidy

causing chromosomal instability.

Along with its roles in chromosomal segregation and mitosis, APC has also been found to

bind the ends of microtubules and stabilize them. It is thought that stabilization of microtubules









by APC occurs through its ability to interact with EB 1 (Morrison et al., 1998; Nakamura et al.,

2001; Su et al., 1995). APC may form a complex with EB 1 and the formin family protein, mDia,

in order to regulate the microtubule network and its stabilization (Wen et al., 2004). Further

evidence is seen in the fact that APC has been found to interact with the microtubule

destabilizing protein kinesin (KIF2C). Moreover, loss of APC causes microtubule

depolymerization to occur much faster than is seen in cells with wild-type APC. Compromised

microtubule stability can lead to failures in mitotic spindle assembly and regulation of the

microtubule network (Kroboth et al., 2007).

Role of APC in cell cycle regulation and apoptosis

Overexpression of APC has been shown to block cell cycle progression from Go/G1 to S phase

by negatively affecting cyclin-cdk complexes. Increased APC has been found to arrest the cell

cycle in the Go/G1-S phase and G2/M phase (Baeg et al., 1995; Heinen et al., 2002). It is thought

that the APC binding partner, human discs large (hDLG) forms a complex with APC that

regulates the G1/S phase transition. APC which lacks the hDLG binding site does not block cell

cycle activity as strongly suggesting that the interaction with hDLG is necessary to down-

regulate cell growth (Ishidate et al., 2000). However, the role of APC in cell cycle control with

damaged DNA is not yet fully understood.

The role of APC in apoptosis is controversial. The precise mechanism is not yet clear. In the

colon, increased levels of APC can induce cell death under certain circumstances. Studies have

been conducted using the HT29 colon cancer cell line which show that expression of full length

APC in this cell line leads to a decrease in cell growth and apoptosis (Morin et al., 1996).

Additional studies with this cell line have shown that expression of full length APC down-

regulated the anti-apoptotic factor Survivin (Zhang et al., 2001). In addition, studies have shown

that APC induces apoptosis by accelerating apoptosis-associated caspase activities (Chen et al.,









2003; Steigerwald et al., 2005). Furthermore, Qian and colleagues have shown that caspase

cleavage of APC and release of its amino terminal domain may be required for APC to accelerate

apoptosis-associated caspase activities independent of transcription. Lastly, some studies have

suggested that a decrease in APC levels is associated with apoptosis. It is possible that both

increased and decreased levels of APC play a role in apoptosis and that different factors such as

cellular signals play a role in whether increased or decreased levels of APC are involved in

apoptosis (Brancolini et al., 1998; Browne et al., 1998; Browne et al., 1994; Jaiswal et al., 2002;

Jaiswal and Narayan, 2004; Schmeiser and Grand, 1999; Williams et al., 1996).

APC and its involvement in DNA damage and repair

The basal level expression of APC occurs through Upstream Stimulating Factors 1 and 2

(USF-1 and USF-2) (Narayan and Jaiswal, 1997). However, APC gene expression is inducible

and upregulated by p53 in response to DNA damage (Jaiswal and Narayan, 2001; Narayan and

Jaiswal, 1997). The presence of damaged DNA usually leads to one of three possibilities. It can

cause the cell to undergo DNA repair, apoptosis, or if the DNA damage persists and the cell

survives it can lead to cell transformation. Since APC gene expression is upregulated in response

to DNA damage, it is possible that APC is playing a role in one or all of these scenarios.

However, it was decided to focus on the involvement of APC in DNA repair since a defect in

DNA repair is one of the initial steps in carcinogenesis. Recently, a DNA Repair Inhibitory

(DRI) domain was identified in APC that allowed it to bind with two base excision repair

proteins DNA polymerase P (pol-P) and Flap endonuclease-1 (Fen-1). Through this interaction it

appeared that APC was able to block pol-P deoxyribose phosphate (dRP) lyase activity,short-

patch base excision repair (SP-BER), pol-P-mediated long-patch base excision repair (LP-BER),

and Fen-1 cleavage activity (Balusu et al., 2007; Jaiswal et al., 2006; Narayan et al., 2005). In









addition to the DRI-domain, the APC protein also contains a mutation cluster region (MCR).

Approximately, 60 percent of the APC mutations in colon cancer arise from the MCR. Mutations

in this region lead to a more severe polyposis than patients with mutations outside the MCR

(Narayan and Roy, 2003). It is of interest to note that the DRI-domain is not in this region,

indicating that both wild-type and truncated APC retain the ability to interact with these proteins

and block BER.

DNA Damage and Repair

The genomes of eukaryotic cells are under constant attack by endogenous and exogenous

sources. Exogenous exposure arises from environmental agents such as ultraviolet (UV)

radiation, ionizing radiation (IR), and various genotoxic agents while endogenous exposure

arises as by-products of cellular metabolism and includes hydrolysis, oxidation, alkylation, and

mismatch of DNA bases (Houtgraaf et al., 2006). Damaged DNA that is not repaired can lead to

genomic instability, apoptosis, or mutagenesis which can eventually lead to carcinogenesis.

Fortunately, the cell has developed various DNA damage checkpoints and DNA repair pathways

to deal with the genomic insults it encounters.

DNA Damage Checkpoints

DNA damage triggers the activation of cell cycle checkpoints. These checkpoints are

activated during the different phases of the cell cycle. The eukaryotic cell cycle contains the

following phases, gap (G)1, synthesis (S), gap (G) 2, mitosis (M) and one phase outside of the

cell cycle Go (Kastan and Bartek, 2004; Nyberg et al., 2002). During G1 the cell becomes larger

and starts synthesizing RNA and proteins which are needed for transcription and translation. The

synthesis of RNA and protein continues through G2. In S phase, the cell replicates its DNA so

that one copy can be passed on to each daughter cell. During G2 the cell continues to grow and

makes extra proteins to ensure that two viable cells can be formed. Lastly, the cell will enter M









phase where the chromosomes will be aligned and two genetically identical daughter cells will

be formed. As long as cells are dividing, the cycle will continue. When cells stop dividing they

are said to be in Go. DNA damage checkpoints generally occur during the transition of each

phase. They cause cell cycle arrest and allow the cell's DNA repair machinery to repair the

damaged DNA. However one checkpoint occurs during a cell cycle phase. The checkpoints are

the G1/S checkpoint, the intra-S checkpoint, and the G2/M checkpoint (Ishikawa et al., 2006).

G1/S checkpoint

In response to DNA damage the G1/S checkpoint serves to prevent the transition between

the G1 phase and the S phase in the cell cycle. Once DNA damage is sensed a myriad of

signaling cascades serve to cause G1 arrest. Briefly, ATM (ataxia-telangiectasia) or ATR (ATM

and RAD related) which are both known as DNA damage sensors, directly phosphorylate the

p53 in its transactivation domain on Ser 15. Moreover, MDM2 is also targeted by ATM/ATR

and CHK2/CHK1 kinases due to its involvement in p53 regulation. Activation of p53 and

MDM2 allows the regulation of the transcription of target genes p21CIP1/WAF l. Once

activated, p21CIP1/WAF 1 inhibits the G1/S cyclin/cdk promoting complexes (Wahl and Carr,

2001). Specifically, the p21CIP1/WAF 1 signal inhibits the RB/E2F pathway and thus prevents

G1/S promotion.

S-phase checkpoint

During the S-phase of the cell cycle DNA replication occurs. In addition to DNA

damage, S-phase checkpoints can be triggered as a result of difficulties with DNA replication

such as stalled replication forks. There are three types of S-phase checkpoints: 1) the replication

dependent checkpoint, 2) the replication independent checkpoint, and 3) the S-M checkpoint.

The replication dependent checkpoint and the S-M checkpoint are dependent upon DNA

replication while the replication independent checkpoint is triggered by double strand breaks. All









three of these checkpoints share components (Bartek et al., 2004). Key components in all three

of the S-phase checkpoints include, RAD 17, and the RAD9-RAD 1-HUS 1 complex, replication

protein A, ATR, the ATR interacting protein (ATRIP), the ATR activator Top BP1, and the

mediator protein CLASPIN. In response to DNA damage all three checkpoints have a similar

response. Once DNA damage is detected, the RAD9-RAD1-HUS 1 complex is loaded onto the

area surrounding the DNA lesion by RAD17. This action allows ATR-mediated phosphorylation

and activation of CHK1 kinase, causing cell cycle arrest (Mordes et al., 2008; Zou et al., 2002).

G2/M checkpoint

The main function of the G2/M checkpoint is to prevent cells with unrepaired DNA

damage from entering mitosis. Cells are prevented from entering mitosis in CHK1 kinase-

mediated manner. Once activated, the G2/M checkpoint begins with ATM/ATR-mediated

phosphorylation of CHKl/CHK2 kinases and p38 kinase, causing degradation, inhibition, or

sequestration of cdc25 phosphatases. Once inhibited, the cdc25 phosphatases can no longer

activate CDK1 which allows the cell to proceed into mitosis. Thus, the cell cycle is arrested

(Nyberg et al, 2002; (Kastan and Bartek, 2004).

DNA Repair

If left unrepaired, DNA damage interferes with replication and transcription leading to

apoptosis. Furthermore, continuous DNA damage can cause accumulation of genetic mutations

that lead to carcinogenesis. However, the cellular system is equipped with repair mechanisms to

deal with damaged DNA. There are six main DNA repair mechanisms or pathways: DNA

damage reversal, nucleotide excision repair (NER), mismatch repair (MMR), homologous

recombination repair (HR), non-homologous end-j oining (NHEJ) repair, and base excision repair

(BER). The type of DNA damage present determines the pathway of DNA repair that is used.









Direct damage reversal

In direct damage reversal, the damage to the base is repaired by methyltransferases or

oxidative demethylases. In general, O-alkylations are repaired by alkyltransferases and N-

alkylations tend to be repaired by oxidative demethylases. These enzymes work by transferring

the alkyl or methyl group from the damaged base onto the enzyme. This action auto-inactivates

the enzyme. Thus, the enzyme is limited by the amount available in the area of the damaged base

(D'Incalci et al., 1988; Gerson, 2004; Middleton and Margison, 2003; Sabharwal and Middleton,

2006).

Nucleotide excision repair

DNA adducts that form large bulky lesions and covalent linkages between adj acent

pyrimidines resulting from UV exposure are repaired through nucleotide excision repair (NER).

NER has two direct pathways termed transcription-coupled repair (TCR) and global genomic

repair (GGR). TCR serves as a system that detects and removes DNA damage that blocks the

progression of RNA polymerase II (Hanawalt, 2002). GGR is slower than TCR and serves as a

system that surveys the entire genome for damage. Both pathways follow a similar process.

Briefly, DNA damage is recognized by the XPC-HR23B complex in GGR and RNA polymerase

II in TCR and the damage is verified by XPA. Subsequently the DNA is unwound by XPB and

XPD helicases that are in a complex with TFIIH basal transcription initiation factor. The DNA is

then incised by XPF and XPG on the damaged strand. Lastly, the damaged portion of the DNA is

removed and the repair is completed by a DNA polymerase followed by DNA ligase. Two

additional proteins are required for TCR, CSB and CSA (Hanawalt, 2002).

Mismatch repair

The mismatch repair (MMR) system removes base-base mismatches and insertion and

deletion loops that are formed during DNA replication (Jiricny, 2006). It is also important in the









maintenance of repeated sequences. Mutations in the MMR system are associated with a pre-

disposition to cancer (Ben Yehuda et al., 2000; Karran, 1996; Skinner and Turker, 2005).

Briefly, base mispairs and small insertion or deletions are recognized and the recognized lesions

are excised. Lastly, the DNA is resynthesized and repair is complete (Neri et al., 2005).

Double-strand break repair

Double strand breaks are repaired by the cell's homologous recombination (HR) and non-

homologous end-joining (NHEJ) repair systems. Repair of these lesions is important because

these lesions can lead to genomic instability, apoptosis and/or mutations due to deletions,

insertions, translocations, and aneuploidy. HR is an error-free repair pathway. In this pathway

homologous sequences are aligned on the sister chromatid (Hefferin and Tomkinson, 2005;

Khanna and Jackson, 2001; Scott and Pandita, 2006). The sister chromatid serves as a template

so that the correct information can be copied to the missing information on the locus where the

break occurred. This process is mediated by RAD5, is aided by proteins in the RAD52 epistasis

group (Helleday, 2003; Jackson, 2002), and usually occurs during the G2 phase of the cell cycle

(Hefferin and Tomkinson, 2005; Khanna and Jackson, 2001; Scott and Pandita, 2006). On the

other hand NHEJ is an error prone process that usually occurs during the Go/G1 phase of the cell

cycle. In NHEJ, the two broken ends are fused together irrespective of homology. The process

begins with Ku70/Ku80 heterodimer binding to the broken ends of the DNA. Then the broken

ends are processed for ligation with the aid of the Artemis-DNA-PKCs complex which is

recruited by the Ku heterodimer. Next, gap filling by a DNA polymerase of the pol-X family

occurs and the resulting nicks are covalently joined by the XRCC4-DNA ligase IV complex

(Lieber, 1999; Lieber et al., 2003). Due to the fact that this process disregards homology, NHEJ

can lead to insertions or deletions of filler DNA making it error prone (Gorbunova and Levy,

1997; Sargent et al., 1997; Seluanov et al., 2004)









Base excision repair

Base excision repair (BER) is a pathway that removes damaged DNA, specifically abasic

DNA (Fig. 1-3). It is composed of two sub-pathways "single nucleotide base excision repair

which is also referred to as short-patch (SP)-BER and "multi-nucleotide base excision repair"

which is also referred to as long-patch (LP)-BER. The pathway used depends on the size of the

repair gap and the type of DNA damage. For example, if the damaged DNA base is reduced or

oxidized it would go through LP-BER (Klungland and Lindahl, 1997; Podlutsky et al., 2001).

In both SP-BER and LP-BER the process begins with the removal of the damaged base. A

DNA glycosylase recognizes the damaged base and cleaves the glycosidic bond between the

sugar and the base leaving an apurinic/apryrimidinic (AP) or abasic site. AP-endonuclease 1

(APE-1) then comes in and makes an incision at the 5'end of the AP-site of the DNA backbone.

The decision is then made whether to use the SP-BER or LP-BER depending on the type of

DNA damage. During SP-BER, the deoxyribose phosphate (dRP) lyase activity of DNA

polymerase P (pol-P), which is located in the 8 kDa domain of pol-P, removes the 5'dRP left

behind by APE-1 resulting in a 5' phosphorylated nicked DNA strand (Matsumoto and Kim,

1995). Pol-P then uses its polymerase activity, which is located in its 31 kDa C-terminal domain,

to synthesize the correct base at the site of the damaged base. The remaining nick is sealed by

DNA ligase I (Mitra et al., 2001).

When the AP-site is oxidized or reduced it becomes chemically resistant to the dRP lyase

activity of pol-P and must be repaired through LP-BER. In this case, pol-P can synthesize up to

13 nucleotides creating a 5' overhang or flap that must be removed to complete LP-BER. The

flap is removed by flap endonuclease-1 (Fen-1) leaving behind a nick which is sealed by DNA

ligase I (Lieber, 1997; Podlutsky et al., 2001). In addition to pol-P, pol-6 and pol-s can also










participate as the main polymerase that synthesizes the 13 nucleotides in strand displacement

synthesis (Fig. 1-3).

Alternate BER pathways. Recently, a different class of sub-pathways of BER have been

discovered. The common denominator in all these sub-pathways is a single strand break. These

pathways have also been termed alternate BER pathways (Almeida and Sobol, 2007). The main

difference in these pathways is in the initiation of BER. The sub-pathways that have been found

are: nucleotide incision repair, NEL2 DNA glycosylase initiated repair, radiation-induced DNA

damage or single strand break repair (SSBR), and strand breaks generated by topoisomerase

poisons. In response to the type of damage that causes the single strand break, one of the sub-

pathways may be initiated (Almeida and Sobol, 2007).

Nucleotide incision repair occurs when APE1 acts directly on the damaged DNA

bypassing the glycosylase step. It makes an incision upstream of the oxidatively damaged base

leaving a 3'OH and 5' phosphate containing the damaged base at the ends. It is thought that this

specifically happens in response to a specific type of oxidative damage, 5-hydroxy-2'-

deoxycytidine (50H-C). The resulting product is a substrate for LP-BER and APE1 goes on to

stimulate Fen-1 activity as well as DNA ligase I activity (Dianova et al., 2001; Ischenko and

Saparbaev, 2002; Ranalli et al., 2002).

NEL2 DNA glycosylase-initiated repair occurs through the use of the bi-functional DNA

glycosylases NEIL1/NEIL2. In addition to removing the oxidized base similarly to mono-

functional glycosylases such as OGG1 and NTH1, NEL1 and NEL2 further process the AP site

to yield a 3' phosphate and 5'phosphate at the margins. Due to the fact that APE l's 3'

phosphatase activity is not very strong, this pathway utilizes another enzyme to process the

resulting NEIL1/NEIL2 product. Instead, polynucleotide kinase phosphatase (PNKP) is required









to remove the 3' phosphate. A complex then forms between NEIL1/NEIL2, pol-P, XRCC1,

DNA ligase III, and indirectly with PNKP. Repair then proceeds in a manner similar to SP-BER

(Caldecott, 2002; Wiederhold et al., 2004).

Radiation-induced DNA damage causes single stranded breaks in the DNA. The pathway

begins with PARP-1 recognizing and binding to single strand breaks. XRCC1 is then recruited

and repair proceeds in the traditional manner of SP-BER. On the other hand, strand breaks

generated by topoisomerase poisons are repaired in a different manner. The lesion in this case

begins as a DNA:topoisomerase 1 (Topl) complex creating a toxic single strand break. The

enzyme tyrosyl DNA phosphodiesterase (Tdp 1) catalyzes the hydrolysis of Topl1 from the DNA

leaving a gap with a 3' phosphate and a 5' hydroxyl. PNKP can then transfer the 3' phosphate to

the 5' end. At this point, gap filling can proceed through pol-P and it is thought that XRCC 1 also

participates (Plo et al., 2003).

APC's proposed role in BER. As previously mentioned it was found that APC was able

to interact with pol-P in vivo and that APC was able to block pol-P-directed strand-displacement

synthesis during LP-BER (Narayan et al., 2005). Furthermore, in 2006, Jaiswal and colleagues

found that APC also interacted with Fen-1 in vivo and in vitro (Jaiswal et al., 2006).

Combined, these findings indicate that APC is playing an important role in modulating BER

activity. Specifically, these data suggest that APC is playing a role in LP-BER.

DNA Polymerase P ( pol-P)

DNA polymerase P is one of the smallest eukaryotic DNA polymerases known to date. It is

one of several known mammalian cellular DNA polymerases. Its maj or role is in DNA repair,

specifically, BER. It has been shown to participate in both SP-BER and LP-BER (Narayan et al.,










2005). Furthermore, it is embryonic lethal indicating that pol-P is necessary for survival (Niimi

et al., 2005).

Pol-j3 Structure

Pol-P is a 39 kDa protein composed of 335 amino acids (Prasad et al., 1998b). It contains

two domains and one protease sensitive hinge region. The first domain is the 8 kDa lyase domain

and the second domain is the 31 kDa polymerase domain. The 8 kDa lyase domain is located at

the amino terminus and contains the lyase activity that is responsible for removing the 5'-

deoxyribose phosphate that is generated after incision of the damaged DNA by APE1 in SP-

BER. The active site of the lyase domain has been identified as Lys72 (Prasad et al., 1998a).

Moreover the residues that form the active site pocket have been identified. The active site

pocket is defined by the following residues: Glu26, Ser30, His34, Lys35, Tyr39, Lys68, Lys72

(Batra et al., 2005; Prasad et al., 2005). The lyase reaction proceeds through a p-elimination

reaction with the active site pocket residues participating in the lyase reaction itself. Tyr3 9 is

proposed to perform nucleophilic attack. Lys35 is proposed to be involved in enzyme mediated

hydrolysis while Glu 26 and Ser 30 are proposed to participate in proton abstraction. However,

the reaction is not well understood (Prasad et al., 2005). In addition to lyase activity, the 8 kDa

lyase domain contains single stranded DNA binding activity (Prasad et al., 1993). The 8kDa

lyase domain is alpha-helical in nature and forms one of two helix-hairpin-helix (HhH) structural

motifs present in pol-P located in residues 55-79 of the lyase domain (Krahn et al., 2004; Mullen

and Wilson, 1997; Pelletier et al., 1996). It has been observed to aid in the stabilization of the 900

bend in nicked DNA and/or in the stabilization of the incised DNA strand by facilitating the

proper positioning of the two free DNA ends (Matsumine et al., 1996; Sawaya et al., 1997).









Like other DNA polymerases, pol-P contains a two metal ion catalytic site found in the 31

kDa polymerase domain which is responsible for the catalysis of the nucleotidyl transferase

reaction (Pelletier et al., 1994; Sawaya et al., 1997). It is located at the C terminus of pol-P. The

31 kDa domain can be broken down into three sub-domains: 1) the D-sub-domain (6 kDa) which

is responsible for dsDNA binding; 2) the C-sub-domain (10 kDa) which houses the catalytic

activity needed for the nucleotidyl transferase reaction; 3) the N-sub-domain (12 kDa) which is

involved in dNTP selection and nascent base pair binding (Beard et al., 2002). The D- and N-

sub-domains are situated on opposite sides of the C-sub-domains (Sobol and Wilson, 2001). The

D-sub-domain contains the other HhH structural motif in pol-P. It is located at residues 92-1 18

(Beard et al., 2002; Krahn et al., 2004; Mullen and Wilson, 1997; Pelletier et al., 1996; Sobol and

Wilson, 2001). The HhH motif serves the same function as the aforementioned HhH motif

located in the 8 kDa lyase domain. The two HhH motifs work together to facilitate the proper

positioning of the two free DNA ends of the DNA substrate. The C-sub-domain possesses

critical amino acids that are important for the catalytic activity of the 3 1 kDa domain. Residues

Aspl190, Aspl92, and Asp256 have been shown to coordinate the two divalent metal cations,

Mg2+ that assist in the nucleotidyl transferase activity and they are known as active site residues

(Date et al., 1991; Menge et al., 1995; Sobol et al., 2000). Two other residues Serl80 and

Argl83 have also been found to play a significant role in catalysis (Date et al., 1990; Kraynov et

al., 2000). The N-sub-domain contains residues involved in pol-P Eidelity. Residues Tyr265 and

Ile260 have been shown to be important in maintaining the fidelity of pol-P. It has been observed

that the N-sub-domain closes around the nascent base pair once the correct nucleotide binds

(Clairmont et al., 1999; Opresko et al., 2000; Opresko et al., 1998; Shah et al., 2001; Shah et al.,

2003; Starcevic et al., 2005). Pol-P is thought to have an induced-fit mechanism for fidelity. In









the induced fit model of fidelity after initial binding of the correct dNTP, the ternary complex

undergoes conformational changes which allow catalytic residues to be aligned for proper

activation of the catalytic site. However, if the incorrect dNTP is incorporated, it is a poor fit and

the alignment of the catalytic residues does not occur correctly which causes sub-par activation

of the catalytic site (Johnson, 1993; Showalter and Tsai, 2002). Additional amino acid residues

shown to be involved in pol-P fidelity are found in a loop under the C-sub-domain. Mutation of

Asp246, Glu249, and Arg253 leads to a decrease in polymerase fidelity (Dalal et al., 2004;

Hamid and Eckert, 2005; Kosa and Sweasy, 1999). Lastly, pol-P contains a protease sensitive

hinge region which connects the 8 kDa lyase domain and the 31 kDa polymerase domain (Prasad

et al. 1998). This region is sensitive to proteases and allows separation of the 8 kDa and 3 1 kDa

domains when it is exposed to proteases.

Roles of Pol-j3

Pol-P plays a dual role in BER. In SP-BER, pol-P uses its lyase activity to remove the 5'-

deoxyribose phosphate (dRP) left behind after APE1 incision (Matsumoto and Kim, 1995). It

then uses its polymerase activity to fill the 1-nt gap left behind with the correct corresponding

nucleotide. While in LP-BER, pol-P uses its polymerase activity to perform strand-displacement

synthesis. Moreover, in studies performed using pol-P deficient cells or in the presence of a

neutralizing antibody, lack of pol-P caused a reduction in DNA repair capacity suggesting a role

for pol-P in LP-BER (Dianov et al., 1999).

Pol-P has been reported to be involved in meiosis and double stranded break repair.

Specifically, pol-P has been shown to be associated in the meiotic events of synapsis and

recombination (Plug et al., 1997). It has also been shown to be involved in non-homologous end

joining in yeast, where Wilson and colleagues discovered a pol-P yeast homologue-dependent










pathway for non-homologous end joining, suggesting that a similar pathway may exist in

mammals although none have been found to date (Wilson and Lieber, 1999).

Post-translational Modifications of Pol-P and its Effect on Pol-P Function

Post-translational modifications are known to affect the activity of the target protein. It

stands to reason that post-translational modifications serve to modulate the desired activity of a

target protein. In the case of pol-P, it would seem that post-translational modification would play

a large role in affecting pol-P's activity with regard to BER. Studies have now confirmed this

thought. Pol-P has been found to undergo acetylation as well as methylation. In 2002, Hasan and

colleagues found that pol-P was able to form a complex with the transcriptional co-activator

p300, and that p300 was able to acetylate pol-P in vitro and in vivo (Hasan et al., 2002).

Interestingly the target for acetylaton by p300 was Lys 72 of pol-P. Lys 72 is the active site for

pol-P lyase activity. Not surprisingly, this acetylation caused a block to pol-P's lyase activity and

it was not able to function properly in a reconstituted BER assay.

More recently, El-Andaloussi and colleagues discovered that Arg 83 and Arg 152 of pol-P

are targets for methylation by protein arginine methyltransferase (PRMT6) in vitro and in vivo

(El-Andaloussi et al., 2007; El-Andaloussi et al., 2006). This methylation stimulates pol-P's

activity. Specifically, it causes an increase in pol-P's polymerase activity but not the lyase

activity. Thus, the methylation of pol-P plays a role in increasing the activity of BER.

Flap Endonuclease 1 (Fen-1)

Fen-1 is a structure specific nuclease that has many roles. It has been found to be involved

in maintaining genome stability through its involvement in DNA replication and repair. It is

embryonic lethal indicating that Fen-1 is critical for survival of the organism. Moreover,

mutations in Fen-1 have been implicated in various diseases including Huntington's disease,










myotonic dystrophy, and cancer (Zheng et al., 2007 [53]). It is thought that Fen-1 may be a novel

tumor suppressor (Liu et al., 2004; Shen et al., 2005; Henneke et al., 2003).

Fen-1 Structure and Biochemical Characteristics

Fen-1 is a 43 kDa protein that houses three domains: The N-domain, I-domain, and C-

domain. The N-and I-domains are important for catalytic activity and are highly conserved

among Fen-1 homologues (Zheng et al., 2002). The C-domain houses the proliferating cell

nuclear antigen (PCNA) motif and the nuclear localization signal and is only found among

higher organisms (Frank et al., 2001; Qiu et al., 2001; Shen et al., 1998). The nuclear

localization signal is critical for eukaryotic organisms because Fen-1 must be in the nucleus to

perform its various roles.

Three individual biochemical activities have been attributed to Fen-1. Harrington and

colleagues discovered the 5' flap endonuclease activity and the 5' to 3' exonuclease activity of

Fen-1 (Harrington and Lieber, 1994). Both of these activities are housed in the same catalytic

domain of the protein. Fen-1 efficiently cleaves DNA or RNA flaps using its 5' endonuclease

activity and this activity is essential for its role in DNA replication where it is involved in

Okazaki fragment maturation (Bambara et al., 1997; Lieber, 1997; Liu et al., 2004). The

exonuclease activity of Fen-1 can cleave nicks, gaps, and 5'end recessed double stranded DNA.

It can also cleave blunt-ended DNA, albeit less efficiently (Lindahl, 1971; Murante et al., 1994).

The role of Fen-1's exonuclease activity is not as well defined, but current thought suggests a

role in Okazaki fragment maturation and apoptosis (Liu et al., 2004; Parrish et al., 2003). The

most recent biochemical activity attributed to Fen-1 is a gap endonuclease (GEN) activity.

Human Fen-1 was found to cleave the template strand of a gapped DNA fork and bubble

substrates which simulate those substrates found in a stalled replication fork (Zheng et al., 2005).









The cleavage was able to occur without the presence of a single stranded 5' DNA flap indicating

that GEN activity and endonuclease activities are separate.

Residues and Stuctural Motifs of Fen-1 Important for Fen-1 Function

Several residues and structural motifs have been identified that are important for the

function of Fen-1 (Fig. 1-4). Amino acids D86, E158, E160, D179, D181 and D233 have been

reported to be critical for the catalytic activity of Fen-1. Mutations of these residues caused a

complete loss of Fen-1 endonuclease activity. The aforementioned residues are thought to be

involved in the coordination of the two Mg2+ Or Mn2+ ions to create the nuclease active site of the

protein (Shen et al., 1997; Zheng et al., 2002). In addition, two more amino acid residues were

identified (K93 and R100) that played a crucial role in catalysis. Mutation of these amino acids

caused a loss of flap endonuclease activity (Qiu et al., 2004). It is thought that these amino acids

may participate in the catalytic reaction itself possibly through activation of a water molecule

that participates in nucleophilic attack.

Fen-1 also contains a H13TH motif which is proposed to mediate the binding of ssDNA and

dsDNA portions of the flap substrate (Hosfield et al., 1998). Other important amino acids

involved in Fen-1 function include: 1) L340, D341, F343, and F344 which are important for the

interaction of PCNA with Fen-1, and 2) K3 54, R3 55, K3 56, K365, K366, and K367 which are

critical for the nuclear localization of Fen-1 in eukaryotic cells (Frank et al., 2001; Qiu et al.,

2001).

Roles of Fen-1

Fen-1 has roles in DNA replication, DNA repair, apoptosis, and in maintaining genome

stability. In DNA replication Fen-1's main role is in Okazaki fragment maturation. In eukaryotic

organisms, DNA synthesis is initiated with short RNA primers that must ultimately be removed.

When the DNA replication proteins encounter a down stream Okazaki fragment, some of the










RNA primer is displaced forming a 5' flap, that must be removed for DNA replication to

continue (Arezi and Kuchta, 2000). The basic thought is that Fen-1 is required to remove or

degrade the RNA primers. Three models have been proposed for Okazaki fragment maturation

and Fen-1 is a major player in all three models. In the first scenario, Fen-1 cooperates with

RNase H to remove the RNA primer leaving a nick which is sealed by DNA ligase I (Turchi et

al., 1994). However, it has been found that RNase H is not required for removal of the RNA

primer (Qiu et al., 1999). Thus, a Fen-1 only pathway has been proposed. In this case, Fen-1

alone removes the entire RNA primer (Murante et al., 1995; Siegal et al., 1992; Turchi and

Bambara, 1993). Lastly, a pathway involving another endonuclease, Dna2p, has been proposed.

In this pathway Dna2p physically interacts with Fen-1 and removes the DNA portion of the RNA

primer flap. Dna2p's activity is stimulated by replication protein A (RPA). However, RPA

inhibits Fen-1 activity. Once cleavage by Dna2p is complete, RPA leaves, and a shorter flap

remains that is cleaved by Fen-1 (Kang et al., 2000).

Fen-1's role in DNA repair has been discussed in the previous section on BER. Briefly,

Fen-1 is required in LP-BER for removal of the flap formed from strand-displacement synthesis.

Moreover, Fen-1 activity in LP-BER is regulated through its interaction with other BER proteins

such as pol-P and PCNA (Gary et al., 1999; Prasad et al., 2000). Additionally, Fen-1 is involved

in another mechanism of LP-BER termed the "Hit-and-Run" mechanism. In this mechanism,

pol-P recognizes and binds to the nicked DNA generated by Fen-1 as a result of the removal of

the flapped DNA. Then pol-P extends the 3'OH displacing the dRP-moiety and fills the 1-nt gap

leaving a nick. Pol-P dissociates from the DNA and Fen-1 binds to the nicked DNA and removes

1-nt using its 5'-3' exonuclease activity. The removal of 1-nt creates a gap and Fen-1 dissociates









from the DNA. This cycle of LP-BER is repeated several times until DNA ligase seals the nick

(Liu et al., 2005).

Interestingly, another role for Fen-1 in DNA repair has been discovered in yeast. Yeast

Fen-1 was found to interact with Pol4 and Dnl4/Lifl, which are a part of the non-homologous

end joining (NHEJ) double stranded break DNA repair pathway (Tseng and Tomkinson, 2004).

Mutations in Fen-1 have been found to increase genomic instability by causing

chromosome loss and accumulation of toxic flaps that may then anneal forming bubbles in the

DNA. Furthermore, it has been shown that abnormal expression of Fen-1 may cause defective

BER and thus, accumulation of mutations that can lead to genomic instability (Liu et al., 2004;

Shibata and Nakamura, 2002). Lastly, another way in which Fen-1 may contribute to genomic

stability is in its role in preventing tri-nucleotide repeat expansion and contraction. Studies in

yeast have shown that mutations in Fen-1 lead to an expansion of tri-nucloeotide repeats through

flap misalignment suggesting that Fen-1 is involved in their prevention (Schweitzer and

Livingston, 1998; White, 1999).

Post Translational Modifications of Fen-1

To date only two types of post translational modifications have been identified for Fen-1:

acetylation and phosphorylation. Both of these modifications have an effect on Fen-1's

endonuclease and exonuclease activities. Acetylation of Fen-1 is accomplished through complex

formation with the transcriptional co-activator p300. Specifically, Lys 354, Lys 375, Lys 377,

and Lys 3 80 are acetylated in vitro and in vivo. Acetylation of Fen-1 causes a reduction in the

endonuclease and exonuclease activities of Fen-1 without affecting PCNA binding. Thus,

acetylation may provide a means of regulating Fen-1 activity and as a result of Fen-1 down

regulation, affecting DNA metabolic events (Hasan et al., 2001; Henneke et al., 2003).










Phosphorylation of Fen-1 also has an effect on its endonuclease and exonuclease activities.

The phosphorylation of Fen-1 is mediated by cyclin dependent kinase (Cdk) 1-cyclin A complex.

Phosphorylation occurs at Ser 187 which is located in the internal nuclease domain. In contrast to

acetylation, phosphorylation abolishes PCNA binding, and thus PCNA-mediated stimulation of

Fen-1 endonuclease and exonuclease activities. However, phopshorylation does not affect the

DNA binding activity of Fen-1. The phosphorylation of Fen-1 by Cdkl-cyclin A suggests a

critical role for phosphorylation as a cell cycle regulatory mechanism for Fen-1 activity

(Henneke et al., 2003).

Purpose/Significance

As previously stated, APC was able to interact with two BER proteins pol-P and Fen-1 and

interfere with pol-P-directed strand-displacement synthesis (Jaiswal et al., 2006; Narayan et al.,

200 5). Some cancer therapeuti c drugs are DNA-alkylating agents such as 1,3 -bi s(2-chl oroethyl)-

1-nitrosourea (BCNU) and temozolomide (TMZ) which create lesions that can be repaired by the

BER pathway. Through a more complete understanding of APC's blockage of BER it may be

possible to improve the efficacy of these chemotherapeutic drugs. Another significance of these

studies is in the basic science aspect of BER. As I discussed above, there are two types of BER

pathways: SP- and LP-BER. How these two pathways are selected by cells is not very clear.

There is some evidence suggesting that the type of DNA damage is responsible for this

distinction. Our studies suggest that APC may play an additional role in distinguishing the SP-

versus LP-BER pathway along with the type of DNA damage present in the cell. By determining

where the interaction of APC with pol-P and Fen-1 is occurring, it will be possible to elucidate

the mechanism that APC is using to block BER. This knowledge will shed more insights into









APC's involvement in BER and how to exploit this involvement to increase the efficacy of DNA

alkylating agents used as chemotherapeutic drugs. This study will focus on determining the

interaction sites of APC with pol-P and Fen-1 with particular emphasis on the APC and Fen-1

interaction and the mechanism by which APC is blocking BER through its interaction with Fen-










Illa~lSLllr~r


t~ilrrr~r


I~r~i~r~


I I


Figure 1-1. Roles of APC. Examples of the many cellular functions that APC has been
implicated in are depicted in the above diagram.










Nuclear asp at signals (IESB)


Oigmerizatairn rpet 15aaIqrepea 2ar epeats
temain domin d I

APC (fullenth) 1

Lt t
MCR I q

1245-KLAAT CKSSNQI...g[EVD FICFSR- 1273-15)


p21 binding


~gregion (?)


Figure 1-2. Structure of the APC protein. Structure of the APC protein showing its p-catenin
binding and downregulation sites, its microtubule binding site, the mutation cluster
region, and the DRI-domain.


reable
tinsite )r
mirrn she














--T-G-a--A--T--C-
--A- C- T -T -A--G-

O ridine DNA Gly~cos~syl

-T-G- -A--T--C- ---TCTC
-A-C-T-T-A-G- -A-C- Ti-A-G-A-G-
oroxdized
APE1 AP site AE


--T--G- OH ~A--T--C-
-A--C- T -T -A-G-


S(dl~iseactivity) d

--T--G-OH P--A--T--C-
--A--C-T--T--A--G-

DNAlipaselw
DNAlipZse IImCCIr~

--T--G-A--A--T-C-
--A--C--T --T --A--G-

91ortpatch BER


-T--OHR -T-C-T-C-
-A-C- T -T -A-G--A--G-

ParxlKNA/RFC
R ~A--A-T--C
--T--G-A--A--T-C T-C-
-A-C- T -T -A-G--A--G-


1 iL~~eFEN i(DEaselD ard PFC

- T -G--A -A -T -C- T -C-
--A--C--T -T --A--G-A--G-


Long-patch BER


Figure 1-3. Base excision repair. BER repair has two main pathways- "Short-patch" BER and
"Long-patch" BER.










Ups resam D]NA


ScrDs tr ale
tl
binding Ieops


~K3;a


ce~ntErT


Figure 1-4. Structure of Fen-1. Diagram depicting important amino acids of Fen-1. Residues in
dark blue show the amino acids involved in the active center of Fen-1. Residues
shown in red are part of the seven loops important for substrate binding while
residues shown in blue are important for post translational modifications. Amino acid
residues shown in yellow are involved in the PCNA interactions while those in green
are involved in nuclear localization of Fen-1. Taken with permission from Shen et al.,
2005.


K254
K200 K252 K267


K128


Nudease











CHAPTER 2
MATERIALS AND METHODS

Yeast Cell Maintenance

The yeast strain PJ694-A was used for all yeast two-hybrid experiments. The yeast strain

PJ69-4A (M~ATa trpl-901 leu2-3,11~2ura3-52 his3-200 gal4A gal80A GAL2-ADE2 LYS2::GAL1-

HIS3 met2::GAL7-lacZ) was grown on synthetic dropout medium lacking lysine [(0.17% Difco

yeast nitrogen base without amino acids, ammonium sulfate (5.0 g/L), complete supplemental

amino acid mixture minus appropriate amino acid containing 2% glucose)]. For long term

storage the yeast cells were maintained in a glycerol stock composed of yeast peptone dextrose

media (YPD) and 40% glycerol and stored at -80oC.

Yeast Two-Hybrid Assays of the Interaction of APC with Pol-j3 and Fen-1

The yeast two-hybrid system was used to determine the functional interaction of APC with

pol-P and Fen-1 in vivo. The APC DNA fragments containing the wild-type (amino acids 1190-

1328) or the DRI-domain mutant (amino acids 1200-1324, in which amino acids Q1256, Il259,

and Y1262 were replaced with alanine) for protein-protein interaction studies were fused to the

yeast Gal4 DNA binding domain (BD) in plasmid pGBDU-C3. Single mutant APC cDNA

fragments of the DRI-domain of APC(Q 1256-A), APC(Il259-A), or APC(Y1262-A) were used

to further characterize the pol-P and Fen-1-interactions and also fused to the yeast Gal4-BD in

plasmid pGBDU-C3. The interacting proteins such as full length pol-P and Fen-1 were fused to

the yeast Gal4 activation domain (AD) in plasmid pGADC3. Adapters were included as needed

for the in-frame insertion of APC, pol-P, and Fen-1 DNA sequences relative to the Gal4-BD or

Gal4-AD plasmids. The yeast strain S. cerevisiae PJ69-4A was co-transformed with pGBDU-C3










and pGAD-C3 derived plasmids and spread on plates containing yeast synthetic dropout (SD)-

UL medium lacking only vector markers Ura for pGBDU-C3 derived plasmids and Leu for

pGAD-C3 derived plasmids. To test for potential protein-protein interactions, transformed cells

were screened for growth on yeast SD-ULH medium, which lacked Ura, Leu, and His, but

contained 5 mM His3 inhibitor, 3-amino-1,2,4-triazole to prevent His3-reporter gene

autoactivation.

Maintenance of Mammalian Cells

Human colon cancer cell lines HCT-116-APC(WT) (wild-type APC expression), HCT116

APC(KD) (knockdown APC expression by pSiRNA-APC) were grown in McCoy's Sa medium,

and LoVo cells (truncated APC expression lacking DRI-domain) were grown in Ham's Fl2

medium at 37 oC under a humidified atmosphere of 5% CO2. In each case, the medium was

supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 Clg/mL

streptomycin.

Generation of Pol-j3 Deletion Constructs

Plasmids were constructed to encode various segments of pol-P (amino acids 60-120, 80-

170, 140-200, and 160-250) to identify the amino acids of pol-P that interact with the DNA

repair inhibitory (DRI) domain of APC. These deletion fragments were subcloned into the

pGAD-C3 vector between the PstI and BamBBBBBBBB~~~~~~~~~HI restriction sites. The following primers were used

to generate various pol-P deletion constructs: pol-P (60-120) (sense primer

5'CGCGGATCCAAGAAATTGCCTGGAGTA-3 and antisense primer 5'

CCAATGCATTGGTTCTGCAGTTTAATTCCTTCATCTAC-3 '), pol-P (80-170) (sense primer

5 '-CGCGGATCCGGAAAATTACGTAAACTG-3 'and antisense primer 5'-

CCAATGCATTGGTTCTGCAGATCCACTTTTTTAACTT-3 '), pol-P (140-200) (sense primer









5 '-CGCGGATCCCTGAAATATTTTGGGGAC-3 and antisense primer 5'-

CCAATGCATTGGTTCTGCAGGAAGCTGGGATGGGTCAG-3 '), and pol-P (160-250) (sense

primer 5'-CGCGGATCCGATATTGTTCTAAATGAA-3 and antisense primer 5'-

CCAATGCATTGGTTCTGCAGATATTCTTTTTCATCATT-3 ').

Site Directed Mutagenesis of Pol-P

Two different sets of pol-P mutants, Set-1 mutant (T79A/K81A/R83A) and Set-2 mutant

(R89A/Q90A/D92A), were generated by use of the Quick Change site-directed mutagenesis kit

from Stratagene (La Jolla, CA). The following primer pairs were used for Set-1 and Set-2

mutants: Set-1, sense primer

5'GAAAAGATTGATGAGTTTTTAGCAGCCGGAGCGTTAGCTAATGAAAT

GGCAG-3' and antisense primer

5'CTGCCGAATCTTTTCCAGTTTAGCTAACGCTCCGGCTGCTAACCTATT

TTC-3'; Set-2, sense primer

5'GGAAAATTACGTAAACTGGAAAAGATTGCCGCGGATGCTAGGTACAC

ATTTCCTG-3' and antisense primer

5'CAGGAAATTGATGGATGAACTCGTAGCATCCGCGGCAAT TTCA TAGA

ATTTTCC-3 '

Yeast Two-Hybrid Assay of Pol-P Deletion Constructs, Pol-P Mutants, and APC

The yeast two-hybrid assay was employed to identify critical amino acids of pol-P to

define the functional interaction with APC in vivo. APC cDNA fragments containing the wild-

type (residues 1190-1328) or the mutant DRI domain (residues 1200-1324, in which amino acids

Ilel259 and Tyrl262 were replaced with alanine) were fused to the yeast Gal4 DNA-binding

domain (BD) in plasmid pGBDU-C3. The interacting pol-P protein fragments such as full-length









and various segments (residues 60-120, 80-170, 140-200, and 160-250) were fused to the yeast

Gal4 activation domain (AD) in plasmid pGAD-C3. Set-1 (T79A/K81A/R83A) and Set-2

(R89A/Q90A/D92A) pol-P mutants also were cloned into plasmid pGAD-C3. Yeast two-hybrid

analysis was performed as described earlier.

APC Peptides

An APC peptide, 20-amino acids in length, (1250-KVS SINQETIQT YCVEDTPI-1269)

was synthesized at the Protein Chemistry and Biomarkers core facility at the Interdisciplinary

Center for Biotechnology Research (ICBR) of the University of Florida. They represent the DRI-

domain of the wild-type APC (APCwt) or a mutated form of the APC, APC(Q-A,I-A,Y-A), or

APC(I-A,Y-A) in which amino acids Q1256, Il259, and Y1262 were replaced with alanine (A)

(1250-KVSSINAETAQTACVEDTPI-1269 or 1250-KVSSINQETAQTACVEDTPI-1269).

Synthesis and Labeling of the Fen-1 Substrate

The Fen-1 substrates for 5'-flap endonuclease activity were made by annealing an

upstream 23-mer (5'-CTAGATGCCTGCAGCTGATGCGC-3') and a downstream 45-mer or

(5' -FTTTTTGTACGGATCCACGTGTACGGTACCGAGGGCGGGTGAC-') or 51-mer

(5 '-AACATTF TTTTTGTACGGATC CACGTGTACGGTACCGAGGGC GGGTC GACA-3 ')

oligonucleotide to a 63-mer complementary template (5' AGATGCCTGCAGCTGATGCG

CGGTACGGATCCACGTGTACGGTACCGAGGGCGGGTCGACA-'). The 45-mer

downstream oligonucleotide has a flap of 5-nts while the 51-mer has a flap of 11-nts. Both flaps

are cleaved by Fen-1. The Fen-1 substrate for 5'-3'exonuclease activity was made similarly

except that the 5-nt flap from the 45-mer oligonucleotides was removed. After annealing with a

63-mer complementary template as described above, these oligonucleotides created a nick in the

DNA suitable for Fen-1 exonuclease activity. The 45-, 51- and 40-mer downstream

oligonucleotides were radiolabeled at the 5'-end with [7-32P]ATP and T4 polynucleotide kinase










(New England Bio Lab, Woburn, MA). The labeled probe was purified by using a nick column

(GE Healthcare, Piscataway, NJ). All three oligonucleotides were annealed at a molar ratio of



Fen-1 Activity in vitro

The assays for 5'-flap endonuclease and 5'-3' exonuclease activities for Fen-1 were

performed in a final volume of 20 C1l. The reaction mixture contained 30 mM Hepes at pH 7.5,

30mM KC1, 8 mM MgCl2, 1 mM dithiothreitol (DTT), 200Clg/mL bovine serum albumin (BSA),

and indicated amounts of Fen-1 and APCwt or DRI-domain mutant APC [APC (I-A, Y-A)]

peptides. After addition of the APC peptides, the reaction mixture was incubated at room

temperature for 5 min. Then 2.5 nM 32P-labeled flapped-DNA or nicked-DNA substrates were

added to the mixture and further incubated at 370C for 30 min. Reactions were terminated with

stop solution containing 0.4% (w/v) SDS and 5 mM EDTA. The DNA was recovered by

phenol/chloroform extraction followed by ethanol precipitation. The 5-nt or 11-nt DNA products

from the 5'-flap endonuclease activity and 1-nt product from the 5'-3' exonuclease activity were

separated on a 15% acrylamide and 7 M urea gel and quantitated by electronic autoradiography

(InstantImager; Packard Instrument Co.,Meriden, CT). Statistical analysis (n=3) was performed

using a t-test on the Sigma Plot computer software.

PCR of Fen-1 Protein Fragments for use in in vitro Transcription/Translation

To identify the APC-binding site on Fen-1, different Fen-1 cDNA constructs were made,

Fen-1 protein fragments were expressed using the in vitro transcription/translation system and

then the interaction of APC and Fen-1 was analyzed by Far-Western Analysis. Primers for each

protein fragment were designed to include a T7 promoter and a kozak sequence in the sense

primer and a stop codon and poly A tail in the anti-sense primer. Five fragments of Fen-1 were










designed (amino acids 1-115, 78-192, 154-268, 231-345, and 269-380). The following primers

were used to generate the protein fragments: Fragment 1-115: (sense primer: 5'

TAATTTTAATACGACTCACTATAGGGAACAGCCACCATGGGATCAGCGC

3' and anti-sense primer:

5'TTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTGAGCCTGCGACGTCCGC

C3'), Fragment 78-192: (sense primer:

5'TAATTTTAATACGACTCACTATGGGAACAGCCACCATGGCTA CCTTT

3' and anti-sense primer:

5'TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATCGCATTACAGCTCACT

3'), Fragment 154-268: (sense primer:

5'TAATTTTAATACGACTCACTATAGGGAACAGCCACCATGGTCCCGGG

CA3' and anti-sense primer:

5'TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAGGGTACTTGGTAGCC

C3'), Fragment 231-345: (sense primer: 5'

TAATTTTAATACGACTCACTATAGGGAACAGCCACCATGGGCGGCATTA

AGT3' and anti-sense primer: 5'

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTTGAAGA TCCAGGCCG

GGT3'), and Fragment 269-3 80: (sense primer: 5'

TAATTTTAATACGACTCACTATAGGGAACAGCCACCATGGTGCGAATGT

CAC3' and anti-sense primer: 5'

TTTTTTTTTTTTTTTTTTTTTTTATTTTCCCCTTTTAAACTTCTCGCCGCT

GC3').









The full length protein was synthesized using the sense primer from 1-115 and the anti-sense

primer from 269-3 80. The PCR of these fragments was carried out using Herculase, a

combination of Pfu DNA polymerase and Taq polymerase, according to the manufacturer' s

recommendations (Stratagene, La Jolla, CA).

Site-Directed Mutagenesis of Fen-1

Two sets of Fen-1 mutants (set 1: K354A/R355A/K356A and set2:

K365A/K366A/K367A) were generated using the Quick Change site directed mutagenesis kit

(Stratagene, La Jolla, CA). To generate each mutant two pairs of primers were used. One pair

was used first to generate two amino acid mutations. Then the resultant mutant protein was used

as template and other primer pair was used to generate the last amino acid mutation. The

following primer pairs were used to generate the set 1 and set 2 mutants: Set-1 primer pair 1,

sense primer: 5'

GGCTCACTCTCTTCAGCTGCGCGCGCAGAGCCAGAACCCAAGGTC' and anti-

sense primer: 5'

GGATCCCTTGGGTTCTGGCTCTGCGCGCGCAGCTGAAGAGAT GC3 and Set-1

primer pair 2, sense primer: 5'

GGCTCACTCTCTTCAGCTGCGGCCGCAGAGCCAGAACCCAAGGTC' and anti-

sense primer: 5'

GGATCCCTTGGGTTCTGGCTCTGCGGCCGCAGCTGAAGAGAGGGC'

Set-2 primer pair 1 sense primer: 5'

GAACCCAAGGGATCCACTGCGAAGGCCGCAAAGACTGGGGCACA' and anti-sense

primer: 5' TGCTGCCCCAGTCTTTGCGGCCTTCGCAGTGGATCCCTTGGGTC'; Set-2

primer pair 2, sense primer: 5'









GAACCCAAGGGATCCACTGCGGCAGCCGCAAAGACTGGGGCACA' and anti-sense

primer5' TGCTGCCCCAGTCTTTGCGGCTGCCGCAGTGGATCCTGTC'

Synthesis of Fen-1 Protein Fragments, Fen-1 wt, and Fen-1 Mutant Proteins Using in vitro
Transcription/Translation

Full-length Fen-1, Fen-1 protein fragments, and Fen-1 set-1 and set-2 mutant proteins

were synthesized using the TNT PCR Quick Kit from Promega (Madison, WI). PCR product was

added directly to the rabbit reticulocyte mix along with the methionine provided with the kit and

50C1M MGl32. The reaction was run as per instructions provided with the TNT PCR Quick Kit

from Promega. Synthesis of Fen-1 proteins and Fen-1 protein fragments was determined by 15%

SDS-PAGE analysis followed by western blotting. Fen-1 proteins and protein fragments were

detected using an anti-mouse Fen-1 antibody (Novus Biologicals, Littleton, CO) that was able to

detect amino acids 1-380 of Fen-1. The signals were detected using the chemiluminescence

technique (GE Healthcare, Piscatway, NJ).

Far-Western Blot Analysis

APCwt and APC(I-A,Y-A) peptides were slot-blotted (0-1 5 Gg) onto a polyvinylidene

difluoride membrane (GE Healthcare, Piscatway, NJ) in a binding buffer containing 20 mM Tris-

Cl at pH 7.4, 100 mM phosphate buffer at pH7.4, 60 mM KC1, and 0.25% (v/v) Nonidet P-40.

After blotting, the membrane was blocked with 5% (w/v) bovine serum albumin and washed

three times with Tris-buffered saline with 0.025% (v/v) Tween-20 prior to incubation with in

vitro transcription/translation synthesized Fen-1 protein fragments (described above), and wild

type, K3 54A/K3 55A/K3 66A mutant or K3 65A/K3 66A/K3 67A mutant human Fen-1 proteins.

Binding was detected using mouse anti-Fen-1 antibody (Novus Biologicals, Littleton, CO). The

signals were detected using the enhanced chemiluminescence technique (GE Healthcare,

Piscatway, NJ).









Pull-Down of Fen-1 wild type and FenS-1 K365A/K366A/K367A from TNT Synthesis
Reaction Mixture

Fen-1 wild-type (Fen-1Iwt) and Fen-1 K3 65A/K3 66A/K3 67A mutant (Fen-1 mutant) were

pulled-down by incubating them with rabbit anti-human Fen-1 antibody in immuno-precipitation

(IP) buffer (30 mM Hepes at pH 7.5, 30 mM KC1, 8 mM MgCl2, 0.01% NP-40, 2% Glycerol) for

4 hours followed by Protein A sepharose beads (Promega, Madison, WI), that were blocked with

5% BSA (v/v), for an additional 2-3 h. Beads were washed with washing buffer (30 mM Hepes

at pH 7.5, 30 mM KC1, 5% glycerol) 3 times and then beads were resuspended in reaction buffer

and used in Fen-1 activity assays. The beads were then used in the indicated amount for the Fen-

1 activity assays.

Immunohistochemistry

Human colon cancer cell lines LoVo, HCT-116-APC(WT), and stable knockdown HCT-

116-APC(KD) were plated into 6-well plates containing sterilized cover slips. Each well

contained about 80-100,000 cells. Cells were allowed to grow for 48 h and were then treated

with 500 CIM MMS for 24 h and fixed using 4% Paraformaldehyde in phosphate-buffered saline

(PB S). For time course experiments, cells were treated with 500 CIM methylmethane sulfonate

(MMS) for 5, 15, or 24 h. At the appropriate time point cells were fixed using 4%

Paraformaldehyde in PBS. Once fixed, cells were quenched using 20 mM ammonium chloride in

PBS. Fixed cells were then incubated in mouse anti Fen-1 antibody (Abcam, Cambridge, MA)

and rabbit anti APC antibody (Santa Cruz, Santa Cruz, CA) for 2hrs. The fixed cells were then

washed four times for 5 min. in PB S and were subsequently incubated in anti mouse Alexa Flour

488 and anti-rabbit Alexa Flour 595 antibodies (Invitrogen, Carlsbad, CA) for 1 h. Cells were

washed as previously described and were mounted onto slides using Vecta Shield (Vector









Laboratories, Burlingame, CA). Slides were viewed under a Zeiss Upright Microscope at

wavelength 500 nm for Fen-1 staining and 640 nm for APC staining.

Western Blot Analysis of Fen-1 and APC Protein Levels in the Cytoplasm and the Nucleus
in Cells Treated with DNA-Alkylating Agents

HCT-116-APC(WT) and HCT-116-APC(KD) cells were treated with 500 CIM

methylmethane sulfonate (MMS) for 5, 10, 15 or 25 h. Nuclear and cytosolic fractions from

MMS-treated HCT-116-APC(WT) and HCT-116-APC(KD) cells were prepared as described in

(Shapiro et al., 1988). In order to determine Fen-1 levels in the nucleus and the cytoplasm, 15 Clg

of the nuclear and cytosolic fractions were analyzed on a 10% SDS-PAGE followed by Western

blotting. A mouse anti-Fen-1 antibody (Novus Biologicals, Littleton, CO) was used to detect the

presence of Fen-1 in the nuclear and cytosolic fractions. The signals were then detected using the

chemiluminescence technique (GE Healthcare, Piscataway, NJ). In order to determine APC

levels in the nucleus and the cytoplasm, 50 Clg of the nuclear fractions and 80 Clg of the cytosolic

fractions were analyzed on a 4% SDS-PAGE followed by Western blotting. The presence of

APC was detected using a mouse anti-APC antibody (Millipore, Temecula, CA). The signals

were then detected using the chemiluminescence technique (GE Healthcare, Piscataway, NJ).









CHAPTER 3
APC INTTERACTS WITH DNA POLYMERASE BETA

Amino acids Thr79/Lys81/Arg83 of pol-j3 are Critical for its Interaction with APC

Previous studies have shown that APC and pol-P interact in vivo and that this interaction

blocks pol-P-directed strand-displacement synthesis (Narayan et al., 2005). In order to map the

critical amino acids of pol-P necessary for its interaction with APC, deletion constructs of pol-

p were generated through PCR amplification and cloned into the pGAD-C3 yeast two-hybrid

(Y2H) vector. A total of four deletion constructs were made (Figure 3-1A). These constructs

were then used in a Y2H assay along with APC wild-type protein (APCwt). As seen in Figure 3-

IB, the deletion constructs containing amino acids 60-120 and 80-170 were able to interact with

APCwt while with the APC DRI-domain mutant [APC(I-A, Y-A)] construct there was no

interaction. Known interactions of wild-type APC and pol-P, PCNA and pol-P were used as

positive controls, while the pol-P-activating domain was used as a negative control for

background colonies. Screening for interacting proteins was performed as described in the

Materials and Methods.

In order to further define the important amino acids from the interacting region of amino

acids 80-120, we examined solvent surface accessibility of the residues implicated in the

interaction with APC. Since a complete structure of APC does not exist, it was not possible to

identify possible interactions through docking modes. Interestingly, amino acids 80-120 are

located in a region that connects the 8 kDa lyase domain and the 31 kDa polymerase domain of

pol-P. From the structure of the ternary substrate complex two regions set-1 (amino acids Thr79,

Lys81, and Arg83) and set-2 (amino acids Arg89, Gln90, and Asp92) were identified that

exhibited high solvent accessibility (Fig. 3-2).









Site-directed mutagenesis was used to change each set of amino acids to alanine and

generate two pol-P mutants. One mutant contains the amino acids of set-1 mutated to alanine and

the other contains the amino acids of set-2 mutated to alanine. The two pol-P mutants were then

cloned into the Y2H vector pGAD-C3 and a Y2H analysis with APCwt was used to determine if

either set of mutants was able to disrupt the interaction between pol-P and APC. As seen in

Figure 3-3, the pol-P set-1 mutant was able to disrupt the interaction while the pol-P set-2 mutant

was unable to interrupt the interaction. This indicates that amino acids Thr79, Lys81, and Arg83

are critical for the interaction of pol-P with APC.

Amino Acids Il259A and Y1262A, but not Q1256A are Required for APC's Interaction
with DNA Polymerase P (Pol-P)

It was originally thought that APC contained a PCNA interacting protein (PIP)-like box

composed of Q1256, Il259, and Y1262 and all three amino acids were necessary for the

interaction of APC with pol-P. In order to test this possibility, site-directed mutagenesis of the

APCwt fragment used previously (Narayan et al., 2005) was carried out. Single mutation of each

amino acid to alanine was carried out and each mutant' s ability to disrupt the interaction of APC

with the proteins was analyzed via Y2H assay (Fig. 3-4). After each single mutant was

constructed, the peptide was cloned into the Y2H vector pGBDU-C3. Full length pol-P was

already in the Y2H pGAD-C3 vector. Screening for interacting proteins was conducted in the

same manner as described in the Materials and Methods. The effects that the single APC mutants

Q1256A, Il259A, and Y1262A had on the ability of APC to interact with pol-P can be seen in

Figure 3-3B. The single mutant Q1256A cannot impede the interaction between pol-P and APC.

However, the single mutants Il259A and Y1262A can disrupt the ability of APC to interact with

pol-P indicating that Y1259A and Y1262A are equally important in the ability of APC to interact










with pol-P. These findings indicate that the PIP-like box found in APC is actually not a PIP-like

box. In order to be a PIP-like box all three amino acids involved must have been involved in the

interaction. Clearly, this is not the case for APC. The PIP-like box was therefore renamed the

DNA Re air Inhibitor (DRI -domain after much consideration. Known interactions of wild-

type APC and pol-P, PCNA and pol-P, and Daxx and p53 were used as positive controls, while

the APC binding domain was used as a negative control for background colonies.










A. Deletion constructs of Pol-p

Pol-p
6010a
80 170 aa

140 200 aa
160 250 aa


335 aa


APC
1190 KAATCKVSSINQETIQTYCVEDTPI 1324 aa
1245 1269""
1259 1262


B. Yeast two-hybrid analysis


Figure 3-1. Interaction of APC with pol-P-deletion constructs. A) Deletion constructs of pol-P
B) Y2H assay of pol-P-deletion constructs and APC; interactions used are depicted in
the diagram. Taken with permission from Balusu et al., 2007.











































Figure 3-2. Ribbon representation of pol-P highlighting the position of key mutants. A) Set 1
(red) and Set 2 (purple) residues are displayed of a ternary substrate complex of pol-
p. Lyase domain is shown in gold and the polymerase domain is shown in blue (PDB
accession code 2MFS). The DNA backbone is shown in orange while a light blue
sphere (Mg2+) identifies the polymerase active site and a red sphere (NZ oflyase 72)
identifies the lyase active site. B) Set 1 (residues 79-84) side chains. C) Set 2
(residues 87-92) side chains. Taken with permission from Balusu et al., 2007.
















Pol-PWt
AEKIDEFLATGKLRKLEKIRQDDTSSS
0 rt f 335 aa
79 81 83 89 90 92
Pol-PMut-1
~ AEKIDEFLAAGALAKLEKIRQDDTSSS
0 f 335 aa
79 81 83
Pol-PMut-2
AEKIDEFLATGcKLRKLEKIAADATSSSS
0 t t 335 aa
8990 92

B. Yeast two-hybrid analysis

1 /2
APC
Pol-pWt
No AP np+
experiment\ Pol-PMut-2





PCNA
+ Pol-PWt
5/ 4


Figure 3-3. Interaction of APC with pol-P mutants. A) Structure of the peptides used in the Y2H
assay. B) Y2H assay of the two pol-P mutants and APC; interactions used are
depicted in the diagram. Adapted with permission from Balusu et al., 2007.


A. Structure of peptides












A. Structure of the APC peptides

APCwt(1190-1328) 1250-KVSSIN QETIQTYCVE DTPI-1269

APC(Q-A,I-A,Y-A) KVSSI NAETAQTACVE DTPI

APC(Q-1256A) KVSSINAETIQTYCVEDTPI

APC(I-1259A) KVSSI NQETAQTYCVE DTPI

APC(Y1262A) KVSSINQETIQTACVEDTPI

B. APC/pol-p interaction

APCwt APC(Q-A,
pol-p Io-A,-A


pol-p wt APC(Q-A)
pol-p

APC(Y-A) APC(I-A)
pol-p pol-B


Figure 3-4. Interaction of APC mutants with pol-P. A) Structure of the peptides used in this
assay. B) Y2H assay of APC mutant proteins with pol-P, interactions used shown in
the diagram. Adapted with permission from Jaiswal et al., 2006.












CHAPTER 4
APC INTTERACTS WITH FLAP ENDONUCLEASE 1 (FEN-1)

APC and Fen-1 interact in vivo

APC and Fen-1 were shown to physically interact by immuno-precipitation and Far-

western analysis (Jaiswal et al., 2006). However, this needed to be confirmed in vivo. In order to

accomplish this, a Y2H assay was used. Full length Fen-1 that was already cloned into the Y2H

vector pGAD-C3, APCwt fragment which contains the DRI-domain (amino acids 1190-1328),

and APC mutant (mut) fragment which contains a mutated DRI-domain (amino acids 1200-1324

with 1259I-A, and 1262Y-A mutations) that was already cloned into the Y2H vector pGBDU-

C3 were used to run the Y2H assay. As Figure 4-1 clearly shows, APCwt is able to interact with

Fen-1 in vivo. The known interaction of PCNA and pol-P was used as a positive control, while

the APC binding domain was used as a negative control to check for background colonies. Lys",

Leu-, Ura-, His-selection media was used to screen for interacting colonies. In order to grow, the

yeast cells needed to make histidine and the only available method to do so is for the two

proteins to interact.

The Interaction Site of APC is the Same for Fen-1 and Pol-j3

Once the interaction site of APC and pol-P was determined, it was thought that the same

amino acids that are necessary for APC's interaction with pol-P may be important for APC's

interaction with Fen-1 since APC interacts with Fen-1 at the DRI-domain. In order to determine

this, a Y2H analysis was carried out with Fen-1 and the single mutants of APC that were

previously used in the yeast two-hybrid analysis with pol-P. It was found that the single mutants

Il259A and Y1262A of APC were able to disrupt the interaction of APC and Fen-1.









However, the Q1256A single mutant of APC did not have an effect on the interaction of APC

and Fen-1 (Fig. 4-2). These results are identical to that of the interaction site of APC and pol-P

indicating that APC interacts with these BER proteins at the same site.

APC Blocks the Endonuclease and Exonuclease Activities of Fen-1

Since APC was found to interact with Fen-1 in vivo and in vitro, the effect of this

interaction on the catalytic activities of Fen-1 was measured. Fen-1 contains two types of

cleavage activity that are relevant for completion of BER: 5' endonuclease activity and 5'-

3'exonuclease activity. The 5' endonuclease activity is important in strand-displacement during

LP-BER where it removes the flap, while the 5'-3' exonuclease activity is important for "Hit-

and-Run" synthesis of LP-BER. In hit-and-run synthesis, Fen-1 excises a nucleotide at the 3'-

side of a nick to create a gap to be filled by pol-P (Klungland and Lindahl, 1997; Prasad et al.,

2001; Qiu et al., 2001; Shibata and Nakamura, 2002). In order to test the effect of APC on Fen-1

cleavage activity, three in vitro assays were run: two which used substrates specific for the 5'

endonuclease activity of Fen-1 and one that used a substrate specific for the 5'-3' exonuclease

activity of Fen-1.

To test APC's effect on Fen-1 5' endonuclease activity, an in vitro 5' flap endonuclease

assay in the presence or absence of APC peptide (20 amino acids long with DRI-domain in the

middle) using a 32P-labeled flapped substrate was conducted. As can be seen in Figures 4-3 and

4-4, cleavage of the substrate by Fen-1 results in a 5 or 11 nt product. When the APCwt peptide

is added cleavage of the flap by Fen-1 is blocked in a dose-dependent manner (compare lane 2

with lanes 3-7 in both Fig. 4-3A and Fig. 4-4A, respectively).









However, when the APC(I-A,Y-A) mutant peptide is used cleavage of the flap by Fen-1 appears

to be unaffected (compare lane 2 with lanes 8-12 in both Fig. 4-3A and Fig. 4-4A, respectively).

A quantitative analysis of the data is shown in Figure 4-3B and Figure 4-4B.

In order to determine the effect of APC on Fen-1 5'-3- exonuclease activity, a nicked DNA

substrate was used. The nicked DNA substrate was formed in the following manner: a 23-mer

upstream and 40-mer downstream oligonucleotides were annealed with a 63-mer complimentary

template oligonucleotide as described in the materials and methods section. The 40-mer

oligonucleotide was labeled with y- 32P[ATP] before annealing at the 5'end. The 5'-3'

exonuclease activity of Fen-1 will cleave the nick resulting in a 1-nt product. The results show a

1-nt cleavage product when Fen-1 is added to the substrate. However, in the presence of APCwt

peptide cleavage of the nicked substrate by Fen-1 is blocked in a dose-dependent manner (Fig. 4-

5A compare lane 2 with lanes 3-6). The 5'-3' exonuclease activity of Fen-1 appears to be

unaffected in the presence of the APC(I-A,Y-A) mutant peptide (Fig. 4-5A compare lane 2 with

lanes 7-10). A quantitative analysis of the data is shown in Figure 4-5B.

Amino Acids K365/K366/K367 are Important for the Interaction of Fen-1 with APC

In order to determine which amino acids of Fen-1 were important for its interaction with

APC, Hyve different protein fragments of Fen-1 were synthesized using the transcription coupled

translation (TNT) Quick PCR kit from Promega (Madison, WI). The Hyve protein fragments are

depicted in Figure 4-6A. Synthesis of the protein fragments was determined via Western blot

analysis. As seen in Figure 4-6B, all protein fragments as well as Fen-1wt were detected. Equal

amounts of proteins were estimated by analyzing the densities of the bands from the Western

blot. A non-specifie band was seen with all expressed Fen-1 fragments, which was originating

due to the cross-reactivity of anti-Fen-1-antibody with TNT extract.









Far-western analysis was then performed using the Fen-1 protein fragments and APCwt

and APC(I-A,Y-A) mutant peptides to determine where the interaction of Fen-1 with APC was

occurring. The only Fen-1 protein fragment able to interact with the APCwt peptide was the Fen-

1 protein fragment which contained amino acids 269-3 80 indicating that this region of Fen-1

might be responsible for the interaction with APC (Fig. 4-7). The overlapping Fen-1 protein

fragment which contained amino acids 231-345 was not able to interact with the APCwt peptide

indicating that the region of Fen-1 that interacts with APC is between amino acids 346-3 80.

The interacting site was further narrowed down by analyzing the role of amino acids 346-

3 80 in the structure and function of Fen-1, and by reviewing the literature for other proteins that

interact with Fen-1 in this region. The most striking thing about amino acids 346-3 80 was that

most of the amino acids are a part of the nuclear localization signal of Fen-1 (Shen et al., 2005).

Furthermore, several proteins have been found to interact with Fen-1 at the C-terminus.

Interestingly, three of the proteins were found to interact with Fen-1 between amino acids 3 53-

3 80 which overlaps with the region of Fen-1 determined to interact with APC from the Far-

western analysis (Guo et al., 2008; Sharma et al., 2005). Based on the above information two

sets of Fen-1 amino acids (K3 54/R3 55/K3 56 and K3 65/K3 66/K3 67) were chosen to mutate to

see if they were important for the interaction of Fen-1 with APC. Each set was chosen based on

its importance in nuclear localization and on the fact that the amino acids chosen were in the

range identified for the interaction of Fen-1 with other proteins such as Werner protein (WRN),

Bloom's syndrome protein (BLM), and endonuclease G (EndoG). Each amino acid was mutated

to alanine and the two Fen-1 mutants were synthesized using the TNT PCR Quick Kit from

Promega (Madison, WI). Synthesis of the Fen-1 mutants was determined using Western blot

analysis. As seen in Figure 4-8, Fen-1wt and mutant proteins were present after synthesis. Equal









amounts of proteins were estimated as described for the Fen-1 protein fragments and the Fen-

1(K3 54A/R3 55A/K3 56A) and Fen-1(K3 65A/K3 66A/K3 67A) mutants were used in a Far-

western analysis. Both Fen-1 (K3 54A/R3 55A/K3 56A) and Fen-1(K3 65A/K3 66A/K3 67A)

mutants were able to decrease the binding of APC and Fen-1. However, the Fen-

1(K3 65A/K3 66A/K3 67A) mutant was able to decrease binding more than the Fen-

1(K3 54A/R3 55A/K3 56A) mutant indicating that the Fen-1(K3 65A/K3 66A/K3 67A) mutant is

more important for binding (Fig. 4-9A). Percent binding of the Far-western analysis can be seen

in Figure 4-9B.

Fen-1(K365A/K366A/K367A) Mutant Retains Catalytic Activity

Due to the fact that the Fen-1(K3 65A/K3 66A/K3 67A) mutant was able to reduce the

binding of APC with Fen-1 and that previous experiments had shown that APC was able to block

Fen-1 catalytic activity, it was decided to investigate whether the Fen-1(K3 65A/K366A/K3 67A)

mutant contained catalytic activity. In order to test the 5'endonuclease activity of the Fen-1

(K365A/K366A/K367A) mutant, a 32P-labeled flapped substrate was used as previously

described in the Results sections. In order to use the TNT synthesized Fen-1wt and Fen-

1(K3 65A/K3 66A/K3 67A) mutant for the assays, the proteins were pulled-down from the TNT

reaction mixture using an anti-Fen-1 antibody. The beads with bound Fen-1wt or Fen-1

(K365A/K366A/K367A) mutant were then used in the endonuclease reaction. Endonuclease

activity of the Fen-1Iwt and Fen-1(K3 65A/K3 66A/K3 67A) mutant was determined by the

presence of a 5-nt product. Interestingly, the Fen-1(K3 65A/K3 66A/K3 67A) mutant was able to

cleave the 5' endonuclease substrate as efficiently as the wild-type protein (Fig. 4-10C compare

lanes 7-9 with lanes 10-12) indicating that mutations in Fen-1 at amino acids K365/K366/K367

are not important for the endonuclease activity of Fen-1.









Additionally, the exonuclease activity of the Fen-1(K3 65A/K3 66A/K3 67A) mutant was

also tested. In order to determine the effect of APC on Fen-1 5'-3- exonuclease activity, a nicked

DNA substrate was used. Labeling of the nicked substrate with y-32P[ATP] was done as

previously described in the Materials and Methods section. Fen-1wt and Fen-

1(K3 65A/K3 66A/K3 67A) mutant proteins were pulled-down from the TNT reaction mixture as

described for the endonuclease activity. The beads with bound Fen-1wt or Fen-

1(K3 65A/K3 66A/K3 67A) mutant were then used in the exonuclease reaction. Exonuclease

activity of the Fen-1Iwt and Fen-1(K3 65A/K3 66A/K3 67A) mutant was determined by the

appearance of a 1-nt product. Surprisingly, the Fen-1(K3 65A/K3 66A/K3 67A) mutant had more

exonuclease activity that the wild-type protein (Fig. 4-10D compare lanes 4 and 5 with lanes 6

and 7). This could be due to the fact that the mutations may have caused a conformational

change within the protein that may have caused the active site to be more accessible. Combined,

the results from the endonuclease and exonuclease assays indicate that the mutations in Fen-1 at

amino acids K365/K366/K367 are not important for the catalytic activity of Fen-1. Therefore,

the blockage of Fen-1 activity by APC after interaction with amino acids K365/K366/K367

could be due to the conformational change of Fen-1 structure affecting its activity. Alternatively,

it could be due to steric hindrance caused by the APC protein since it is such a large protein.

APC Prevents the Entry of Fen-1 into the Nucleus after Exposure to DNA-Alkylating
Agents

The fact that the Fen-1(K3 65A/K3 66A/K3 67A) mutant still contained catalytic activity

prompted the idea that APC could be blocking Fen-1's participation in LP-BER through more

than one mechanism. Since the Fen-1(K3 65A/K3 66A/K3 67A) mutant does not affect catalytic

activity, the purpose of an interaction with APC at this site remained unclear. In order to get a

better idea of the functional interaction of APC with Fen-1 at amino acids K3 65/K3 66/K3 67 an









immunohistochemical analysis was performed to determine the localization of these two proteins

in cells after exposure to the DNA-alkylating agent, methylmethane sulfonate (MMS). In 2007,

Kundu and colleagues, showed that the treatment of cells with 500 CIM of MMS caused the

upregulation of APC and that this upregulation of APC caused a blockage of DNA repair. Based

on these data, HCT-116-APC(WT) cells were treated with 500 CIM MMS to ensure that APC

upregulation would occur and that it would interact with Fen-1. HCT-116-APC(WT) cells

contain wild-type APC. Particular attention was paid to the localization of APC and Fen-1 with

regards to the nucleus as DNA repair occurs in the nucleus and the identified APC interaction

site on Fen-1 occurs in the nuclear localization signal. After 24 h of treatment with 500 C1M

MMS, an interesting phenomenon can be seen in the HCT-116-APC(WT) cells. Fen-1 nuclear

localization is severely decreased while the cytoplasmic level of Fen-1 has increased as

compared to control HCT-1 16-APC(WT) cells. Moreover, when the staining of Fen-1 (green)

and APC (red) is merged a considerable amount of co-localization, as demarcated by a yellow

color, is seen between Fen-1 and APC in the cytoplasm and more specifically around the nuclear

membrane as compared to control HCT-116-APC(WT) cells (Fig 4-11). Interestingly, this

phenomenon was not seen in HCT-1 16-APC(KD) cells subj ected to the same treatment as the

HCT-116-APC(WT) cells. The HCT-116-APC(KD) cell line has been stably transfected with

siRNA against APC. Thus, APC levels are greatly reduced in this cell line. As seen in Figure 4-

12, after 24 h of treatment the HCT-1 16-APC(KD) cells still contain the maj ority of Fen-1 in the

nucleus indicating that APC's interaction with Fen-1 may be playing a role in the loss of nuclear

Fen-1 in response to treatment with MMS.

In order to gain a more complete understanding of what was occurring during the 24 h

treatment with MMS, a time course experiment was performed. The cells were treated with 500










CIM MMS for a time period of 5, 15, or 24 h. In addition to the aforementioned cell lines HCT-

116-APC(WT) and HCT-116-APC(KD), the LoVo cell line was also used. LoVo cells were used

because they contain truncated APC (120 kDa) which lacks the DRI-domain and will not be able

to interact with Fen-1. Thus, these cells serve as an additional method to ensure that the loss of

nuclear Fen-1 after treatment with MMS for 24 h is dependent upon the APC and Fen-1

interaction. As expected, both LoVo cells and HCT-116-APC(KD) cells retained nuclear Fen-1

throughout the length of the time course experiment (Fig. 4-12 and 4-13). Moreover, co-

localization (yellow) was not seen in the merged photographs of APC (red) and Fen-1 (green).

On the other hand, a significant difference was observed between HCT-116-APC(WT) cells

treated with MMS versus control cells as the time course experiment progressed. After 5 h of

treatment, Fen-1 (green) is still localized in the nucleus and some Fen-1 is present in the

cytoplasm while, APC (red) is seen in both the cytoplasm and the nucleus (Fig. 4-13). When the

localization of APC (red) and Fen-1 (green) are merged, more co-localization (yellow) is seen in

both the cytoplasm and the nucleus of the HCT-1 16-APC(WT) cells as compared to control cells

(Fig. 4-14), indicating that in response to DNA damage there is an increased interaction between

Fen-1 and APC. After 15 h of MMS treatment, about 50 percent of cells have developed a loss of

nuclear Fen-1 (green) and an increase of cytoplasmic Fen-1 as compared to cells treated for only

5 h and HCT-116-APC(WT) control cells. Furthermore, APC (red) is localized more in the

cytoplasm than in the nucleus. When APC (red) and Fen-1 (green) localization is merged a

considerable amount of cytoplasmic co-localization (yellow) is seen. Interestingly, co-

localization can also be seen in the nucleus where the remaining Fen-1 is present (Fig. 4-12).

After 24 h of treatment, most of the cells have developed a loss of nuclear Fen-1 (green) and an

increase in cytoplasmic Fen-1 as compared to control cells and cells treated with MMS for only 5









h. Furthermore, most of the APC (red) appears to be localized in the cytoplasm as compared to

the nucleus although some nuclear APC also remains in HCT-116-APC(WT) cells (Fig. 4-12).

Lastly, when the localization of Fen-1 (green) and APC (red) is merged a significant amount of

co-localization (yellow) is seen in the cytoplasm especially around the nuclear membrane.

Moreover, some co-localization can still be seen between the remaining nuclear Fen-1 and APC,

which is much lower than the interaction between Fen-1 and APC which are co-localized in the

cytoplasm after 24 h of MMS treatment (Fig. 4-12). Thus, it appears that APC is required for the

loss of nuclear Fen-1 over time since neither LoVo cells nor HCT-1 16-APC(KD) cells exhibited

this phenomenon (Fig. 4-12 to 4-13). This discovery provides a role for the interaction of APC

with Fen-1 at its nuclear localization signal by suggesting that APC prevents Fen-1 from entering

the nucleus after exposure to DNA-damaging agents.

In order to confirm the immunofluorescence results, nuclear and cytosolic levels in HCT-

116-APC(WT) and HCT-116-APC(KD) cells treated with 500 CIM MMS for 5, 10, 15, and 25 h

were determined by Western blot analysis. The cytosolic fraction showed no significant change

in the levels of Fen-1 in both HCT-1 16-APC(WT) cells and HCT-1 16-APC(KD) cells treated

with 500 CIM MMS (Fig. 4-14) while the nuclear fraction showed a decrease in the levels of

Fen-1 in MMS treated cells over time in HCT-116-APC(WT) when compared to control HCT-

116-APC(WT) cells (Fig. 4-14). However, no significant change was seen in the levels of

nuclear Fen-1 in MMS treated HCT-116-APC(KD) cells (Fig. 4-15). These results are consistent

with the findings of the immunofluorescence study, which showed similar results for nuclear

Fen-1. Analysis of the APC levels in the cytosolic and nuclear fractions showed an increase in

the level of APC in MMS-treated HCT-1 16-APC(WT) cells over time. As expected, APC levels

were undetectable in MMS treated HCT-116-APC(KD) cells in both the nuclear and cytosolic









fractions. Furthermore, the increase in cytoplasmic APC correlates with the decrease in nuclear

Fen-1 suggesting that the cytoplasmic APC is preventing Fen-1 translocation into the nucleus. In

HCT-116-APC(WT) cells treated with MMS for 15 h cytoplasmic APC reaches its highest levels

while nuclear Fen-1 reaches its lowest levels. At the 25 h time point, the cytoplasmic APC

begins to decrease while nuclear Fen-1 shows a modest increase which further supports the

hypothesis that APC may be blocking nuclear translocation of Fen-1 in response to DNA-

alkylating agents.














9 1328 aa



9 1324 aa


Full-length Fen-1
D 380 aa


A. Structure of the Peptides


APCwt
~ KAATCK~VSSINQETIQTYCVEDTPI
1190 1245 126!

APC(I-A,Y-A)
------KAATCKVSSI NQETAQTACVE DTPI
1200 1245 126!


B. Yeast-Two Hybrid Analysis


Figure 4-1. Interaction of Fen-1 and APC. Y2H assay showing the in vivo interaction of APCwt
and Fen-1wt. A) Structure of the peptides used in the Y2H assay. B) Y2H assay of
APC and Fen-1. Interactions used are shown in the diagram.









A. Structure of the APC peptides

APCwt(1190-1328) 1250-KVSSINQETIQTYCVEDTPI-1269

APC(Q-A,I-A,Y-A) KVSSINAETAQTACVEDTPI

APC(Q-1256A) KVSSINAETIQTYCVEDTPI

APC(I-1259A) KVSSINQETAQTYCVE DTPI

APC(Y1262A) KVSSINQETIQTACVEDTPI


B. APC/Fen-1 interaction
APCwt IAPC(Q-A,
Fen-1 I-'A,Y-A)
Fen-1

IAPCwt \ APC(Q-A)
Fen-1

p53 APC(I-A)
DAXX Fen-1

PCNA APC(Y-A)
pol-B Fen-1


Figure 4-2. Interaction of APC mutants with Fen-1. A) Structure of the peptides used in this
assay. B) Y2H assay of APC mutant proteins with Fen-1, interactions used shown in
the diagram. Adapted with permission from Jaiswal et al., 2006.





1 2 3 4 5 6 7 8 9 10 11 12
- 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
-0.2 0.5 1.0 1.6 2.0 -
- 0.2 0.5 1.0 1.6 2.0

analysis


. I I I I I I , ,


-0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
-- 0.2 0.5 1.0 1.6 2.0 -
- 0.2 0.5 1.0 1.6 2.0


A. Endonuclease activity 45-mer


23-mer


3+P-labele DmN


5-nt products --)


Fen-1 (nM)
APCw~t (pLM)
APC(I-A,Y-A) (pIM)

B. Quantitative
50 I-


*p-value<0.05
-~ **p-value<0.001







-~I


I)
I)
I)


01

Fen-1 (nM
APCwt (pM
APC(I-A,Y-A) (I M


1 2 3


4 5 6 7 8 9 10 11 12


Figure 4-3. APC blocks the 5'-flap endonuclease activity of Fen-1. A) Autoradiogram of a
representative experiment. Reaction mixtures in a 25 CIL final volume contained a
fixed (0.1 nM) concentrations of Fen-1 coupled with different concentrations (0.2,
0.5, 1.0, 1.6, and 2.0 CIM) of either APCwt or mutant APC(I-A,Y-A) peptides. The
mixture was incubated for 5 min on ice, and then 2.5 nM of the 32P-labeled flapped-
DNA substrate was added. The cleavage reaction was carried out at 37oC for 30 min.
X-ray film autoradiography determined the 5-nt cleaved product, and the electronic
autoradiography measured the radioactivity (InstantImager; Packard Instrument Co.,
Meriden, CT). B) Quantitative analysis of the 5-mer cleaved product. The fraction of
cleavage was calculated as the percent radioactivity present in the cleaved product as
follows: % cleavage = [5-nt/(45-mer + 5nt)]-100. Data are the mean & SE of three
different experiments.











1


B. Quantitative analysis


1 2 3 4 5 6 7 8 9 10 11 12


., Endonuclease activity so-mer


23-mer
,,~ji63-mer


32P-labele N


11-nt products ~-





Fen-1 (nM)
APCwt (pN)
APC(I-A,Y-A) (IM)


- 0.10.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.10.1
-- 0.2 0.5 1.0 1.6 2.0- --
- 0.2 0.5 1.0 1.6 2.0


1 2 3 4 5 6 7 8 9 10 11 12
Fen-1 (nM) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
APCwt (pM) 0.2 0.5 1.0 1.6 2.0 -
APC(I-A,Y-A) (pM) 0.2 0.5 1.0 1.6 2.0


Figure 4-4. APC blocks the 5'-flap endonuclease activity of Fen-1 regardless of flap size. A)
Autoradiogram of a representative experiment. Reaction mixtures in a 25 CIL final
volume contained a fixed (0. 1 nM) concentrations of Fen-1 coupled with different
concentrations (0.2, 0.5, 1.0, 1.6, and 2.0 CIM) of either APCwt or mutant APC(I-
A,Y-A) peptides. The mixture was incubated for 5 min on ice, and then 2.5 nM of the
32P-labeled flapped-DNA substrate was added. The cleavage reaction was carried out
at 37oC for 30 min. X-ray film autoradiography determined the 5-nt cleaved product,
and the electronic autoradiography measured the radioactivity (InstantImager;
Packard Instrument Co., Meriden, CT). B) Quantitative analysis of the 11-mer
cleaved product. The fraction of cleavage was calculated as the percent radioactivity
present in the cleaved product as follows: % cleavage = [11-nt/(45-mer + 11-nt)] -
100. Data are the mean & SE of three different experiments.


A_ _



























1 2 3 4 5 6 78 9 10


analysis


*p-value<0.05












1 2345678910


A. Exonuclease activity
23-mer
63-mer
63-mer
3zP-labeled DNA





1-nt products


Fen-1 (nM)
APCwt (CtM)
APC(E-A,Y-A) (CtM)

B. Quantitative
so .


15 15 15 15 15 15 15
3.0 5.8 7.8 8.8 -
- -3.0 5.8


15 15

7.8 8.8


Fen-1 (nM)
APCwt (ptM)
APC(I-A,Y-A) (CtM)


S15


15 15 15 15
3.0 5.8 7.8 8.8


15 15 15 15

3.0 5.8 7.8 8.8


Figure 4-5. APC blocks the 5'-3' exonuclease activity of Fen-1. Exonuclease activity reaction
mixture was assembled in a total of25 CIL final volume and contained fixed (15 nM)
concentrations of Fen-1 along with (3, 5.8, 7.3 or 8.8 C1M of either APCwt or APC(I-
A,Y-A) peptides. The mixture was incubated for 5 min at 220C and then 2.5 nM 63-
mer oligonucleotide substrates containing the 32P-labeled 40-mer downstream
oligonucleotides were added. The exonuclease reaction was carried out at 370C for 30
min. A) X-ray autoradiography determined the 1-nt cleaved product. B) Quantitative
Analysis using electronic autoradiography to measured the radioactivity.
(InstantImager; Packard Instrument Co., Meriden, CT). The fraction of cleavage was
calculated as the percent radioactivity present in the cleaved product as follows: %
cleavage = [1-nt/(40-mer + 1-nt)]-100. Data are the mean & SE of three different
experiments.











A. Structure of the protein fragments

Full length Fen-1


380 aa


1 115 aa

78 192 aa

154 268 aa

231~ 345 aa

269 380 aa

B. Western blot


MW
45 kDa -




14 kDa -



Control rabbit reticulocyte
Fen-1wt
Fen-1(1-115)
Fen-1(78-192)
Fen-1(154-268)
Fen-1(231-345)
Fen-1(269-380)


e-43 kDa
e- NS



]12-14 kDa


123 4 5 6 7
+ - -
- + - -
- + -
- + -
- + -
- - + -
- - +


Figure 4-6. Protein levels of in vitro transcription/translation of wild-type and fragments of Fen-
1. A) Depiction of protein fragments of Fen-1. B) TNT synthesis of Fen-1 wild- type
and Fen-1 protein fragments. Proteins and protein fragments were run on a 15% SDS-
PAGE followed by Western blot analysis. Fen-1 wild-type protein and protein
fragments were detected using mouse anti-Fen-lantibody from Novus Biologicals.









Fen-1(1-1151


Fen-1(78-1921


Fen-1wt


*0







O





~10


u 2.
(


O




2.5

5

10


APC APCwt
:I-A,Y-A)


APC APCwt
(I-A,Y-A)





Fen-1(231-345)


APC APCwt
(I-A,Y-A)


Fen-1(154-268)


Fen-1(269-380)


APC APCwt APC APCwt
I-A,Y-A) (I-A,Y-A)


APC
(I-A,Y-A)


APCwt


Figure 4-7. Far-western analysis of the interaction of APCwt and APC(I-A,Y-A) peptides and
Fen-1 protein fragments. APC peptide was immobilized onto a PVDF membrane and
incubated with either Fen-1 wild type or the indicated Fen-1 protein fragment.
Binding was detected through the use of a mouse anti-human Fen-1 antibody
followed by chemiluminescence analysis. Fen-1 protein fragment 269-3 80 was the
only fragment able to interact with APCwt.










A. Structure of wild-type and mutant Fen-1 proteins


Fen-1wt
LSSAKRKEP -STKKKAKT
1 358 350 363 370 380 aa

Fen-1(K354A/ R355A/ K356A) mutant
LSSAAAAEP -STKKKAKT
1 358 350 363 370 380 aa

Fen-1(K365A/ K366A/ K367A) mutant
LSSAKRKEP -STKKKAKT
1 358 350 363 370 380 aa

B. Western blot


45 kDa -4ka





1 2 3
Fen-1(wt) +--
Fen-1(K354A/ R355A/K356A)- +-
Fen-1(K365A/ K366A/K367A)-- +

Figure 4-8. Protein levels of in vitro transcription/translation of wild-type and mutant Fen-1. A)
Depiction of Fen-1 mutant proteins. B) Western blot Analysis Depicting Result of
TNT Synthesis of Fen-1 wild-type and mutant Proteins. Fen-1 wild-type and mutant
proteins were synthesized using the TNT PCR Quick Kit (Promega, Madison, WI).
Synthesized proteins were run on a 10% SDS-PAGE and then transferred onto a
PVDF membrane. Membrane was probed for Fen-1 wild-type and mutant proteins
using a mouse anti-human Fen-1 antibody and the signal was detected using
chemiluminescence analysis.

























B. Quantitative analysis
120 a


0~~0~----- 0~~0~----- 0~~0~-----


A. Far-Western blot


Fen-1 Fen-1
Fen-1wt
(K354A/R355A/K356A)(K365A/K366A/K367A)


o


a.1
O 5
(I


APC APCwt
-A,Y-A)


APC APCwt
(I-A,Y-A)


APC APCwt
(I-A,Y-A)


Fen-1wt


Fen-1(K354A/
K355A/K356A)
I I


Fen-1(K365A/
K366A/K367A)
I I


too


O L
APCwt (C19)


APC(I-AY-A) (cl9)-----oL"L"OL"
r3 ''''


-----OLnLnOLn


-----OLnLnOLn


Figure 4-9. Far-western analysis of the interaction of Fen-1 wild-type and mutant proteins with
APC. A) APCwt and mut peptides were immobilized on a PVDF membrane and
incubated with Fen-1 wild-type and mutant proteins. Binding was detected using a
mouse anti-human Fen-1 antibody followed by chemiluminescence. B) Percent
binding of APCwt and Fen-1 wild-type and mutant proteins. Percent binding was
determined by using the density of APC and Fen-1 wild-type binding band as 100 %
binding and comparing Fen-1 mutant proteins and APC-binding to the density of the
APC and Fen-1 wild-type band [(Density of APCwt and Fen-1 mutant
binding/Density of APCwt and Fen-1 wild-type) x 100 = Percent bound)].
Experiment was repeated twice.


nihnn.


n~s,, Tili~a











A. Protocol B. Western blot
Synthesis of
protein by TNT Fe1


Pull-down with
anti-Fen-1 antibody
12 345

V1 Reticulocyte controluiidFn l +g -
Fen-1 bound with beads Fen-1wt --+--
was used for endo- and Fe nl(K365A/ K366A/ K36 7A) ---+-
exonuclease assays Bead Control ----+

C. Endonuclease activity D. Exonuclease activity
45-mer

23-mer 23-mer
63-mer 63-mer
63-mer 4
32P-labeled
DNA

5-nt
products m me o o m m c pdut
Purified Fen-1 (nM) .1 .2 - - -- - 10
Reticulocyte control (pl) 2 4 6 - 3 6-----
Fen-1wt (pl) - 2 4 6 -- 3 6---
Fen-1(K365A/K266AJ - - 2 4 6 - 3 6-
K367A) (plI)


Figure 4-10. Fen-1 (K365A/K366A/K367A) mutant retains 5'-flap endonuclease and 5'-3'
exonuclease activity. A) Pull-down assay of wild-type and mutant Fen-1 proteins.
Fen-1 wild type and Fen-1 (K365A/K366A/K367A) mutant protein were synthesized
using the TNT PCR Quick Kit (Promega, Madison, WI) and then pulled down from
the TNT reaction mixture using protein A sepharose beads and rabbit anti human
Fen-1 antibody (Bethyl laboratories, Montgomery, TX). Beads were used in western
blot analysis to detect presence of pulled-down Fen-1(wt) and Fen-
1(K3 65A/K3 66A/K3 67A) mutant protein. Proteins were detected using anti-Fen-1
antibody (Novus Biologicals, Littleton, Co) and the chemiluminesence technique (GE
Healthcare, Picastaway, NJ) B) Schematic of protocol used for endonuclease and
exonuclease assay. C) Autoradiogram depicting endonuclease activity. Reaction
mixtures in a 25 CIL final volume contained 2, 4, or 6 Cll of beads with Fen-1 wild-
type or Fen-1(K3 65A/K3 66A/K3 67A) mutant protein. Purified Fen-1 wild-type
protein at a concentration of 0. 1 or 0.2 nM was used as a positive control. The
mixture was incubated for 5 min on ice, and then 2.5 nM of the 32P-labeled flapped-
DNA substrate was added. The cleavage reaction was carried out at 37oC for 30 min.
X-ray film autoradiography determined the 5-nt cleaved product, and the electronic
autoradiography measured the radioactivity (InstantImager; Packard Instrument Co.,
Meriden, CT). D) Autoradiogram depicting exonuclease activity. Reaction was
carried out as described in B with the following changes: 1. 3 or 6 Cll of beads with
Fen-1 wild-type or Fen-1(K3 65A/K3 66A/K3 67A) was used in the reaction mixture;
2. 10 nm of purified Fen-1 wild-type protein was used as a positive control; 3. A 32P-
labeled nicked-DNA substrate was used; 4. X-ray film autoradiography determined
the 1-nt cleaved product and electronic autoradiography measured the radioactivity.









DAPI,
Alexa-594
merged


Alexa-594,
Alexa-488
merged


Alexa -594
(APC)


Alexa-488
(Fen-1)


DAPI


Figure 4-11. Imunohistochemical analysis of APC and Fen-1 co-localization in HCT-1 16-
APC(WT) and HCT-116-APC(KD) cells. Cells were treated with the indicated
amount of MMS for 24 h and then stained for Fen-1 (green), APC (red), and nuclei
(blue). Images were merged to determine co-localization.





A. HCT-116-APC(WT) cells B. HCT-116-APC(KD) cells
DAPI, DAPI,
Alexa-594 Alexa-594, Alexa-594 Alexa-594,
Alexa-594 Alexa-488 Alexa-488 Alexa-488 Alexa-594 Alexa-488 Alexa-488 Alexa-488
(APC) (Fen-1) DAPI merged merged (APC) (Fen-1) DAPI merged merged

11118gS HMHMH


1111551 HM HH1
v,11111 Hr 111M


11 HMHMH11

Figure 4-12. A time-course analysis of co-localization of APC and Fen-1 in colon cancer cells
treated with MMS. A) HCT-116-APC(WT) cells were treated with 500 CIM for the
indicated amount of time. Cells were stained for Fen-1 (green), APC (red), and nuclei
(blue). Pictures were then merged for co-localization. B) HCT-116-APC(KD) cells.
Cells were treated the same as HCT-116-APC(WT).





DAPI,
Alexa-594 Alexa-594,
Alexa-488 Alexa-488
meraed meraed


Alexa-594 Alexa-488
(APCI (Fen-11


DAPI


MMMM
HMHM
HMHM


H
H
5


Figure 4-13. A time-course analysis of co-localization of APC and Fen-1 in LoVo cells treated
with MMS. LoVo cells (truncated APC that lacks the DRI-domain) were treated with
500 C1M MMS for the indicated amount of time. Cells were stained for Fen-1 (green),
APC (red), and nuclei (blue). Pictures were then merged for co-localization.


ILoVo cells










A. Cytosolic fraction


APC -


p-Actin ,~ 1C

Time(h) o mo mmulo mom ulu

HCT-116-APC(WT) + + + + + -----


+ + + + + - -


HCT-116-APC(KD) ---


-+ ++ + +


-++ ++ +


MMS (500 CM)-+ + ++ -++ + +


-+ + ++ + +++


Figure 4-14.A time-course analysis of nuclear and cytosolic protein levels of APC and Fen-1 in
MMS-treated colon cancer cells. HCT-116-APC(WT) and HCT-116-APC(KD) cells
were treated with 500 CIM MMS for the indicated amount of time, Nuclear and
cyosolic fractions were prepared and analyzed using western blot analysis. Fen-1 was
detected using mouse anti-Fen-1 antibody (Novus Biologicals, Littleton, Co) while
APC was detected using mouse anti-APC antibody (Millipore, CA). Signals were
detected using the chemiluminescence technique. A) Western blot analysis of APC
and Fen-1 levels in the cytosolic fractions of MMS treated HCT-1 16-APC(WT) and
HCT-116-APC(KD) cells. p-actin is shown as a loading control. B) Western blot
analysis of APC and Fen-1 levels in the nuclear fractions of MMS treated HCT-1 16-
APC(WT) and HCT-116-APC(KD) cells.


B. Nuclear fraction









CHAPTER 5
DISCUS SION

The Interaction of APC and DNA Polymerase |3 and its Implications in BER

In the present study the interaction sites of pol-P and APC involved in the APC and pol-P

interaction were determined. Using Y2H analysis with single amino acid mutants of APC, it was

found that only Il259 or Y1262 were necessary for the interaction of APC with pol-P.

Furthermore, the mutation of either amino acid to alanine was enough to disrupt the interaction

of APC with pol-P suggesting that the interaction of pol-P may be at the surface instead of in the

groove of the APC protein. Additionally, the present study indicates that APC interacts with a

specific region of pol-P. Based on the Y2H analysis with pol-P-deletion constructs and APC, it

was determined that amino acids 80-120 of pol-P were critical for the interaction of pol-P with

APC. Upon analysis of the crystal structure of pol-P and examining the solvent accessibility of

amino acids 80-120, possible amino acids involved in the interaction with APC were determined.

Mutagenesis of Thr79, Lys81, and Arg83 to alanine abolished the interaction between pol-P and

APC. Thus, amino acids Thr79, Lys81, and Arg83 were identified as critical residues of pol-P

which were important for the interaction with APC. Notably, these amino acids are located in the

linker region of pol-P which connects its lyase and polymerase domains (Beard and Wilson,

2006).

The active site of the lyase domain of pol-P is Lys72 which is closely located to the amino

acids Thr79, Lys81, and Arg83 (Srivastava et al., 1998). Due to this close proximity, it would be

logical to hypothesize that the binding of APC can interfere with pol-P's deoxyribose phosphate

(dRP)-lyase activity, and thus SP-BER. In fact, studies conducted indicate that this hypothesis is

correct (Balusu et al., 2007). By reconstituting the SP-BER system in vitro, these authors showed









that SP-BER was blocked by APC in a dose-dependent manner. Furthermore, they determined

the mechanism by which APC is blocking SP-BER by testing whether APC blocked pol-P dRP-

lyase activity. The dRP-lyase activity of pol-P has been determined as the rate-limiting step in

SP-BER. Removal of the 5'dRP moiety is crucial for the generation of the 5' phosphate required

by DNA ligase to seal the nick and complete repair of the DNA (Beard and Wilson, 2006;

Srivastava et al., 1998). Through the use of an in vitro dRP-lyase activity assay Balusu and

colleagues were able to examine the effect of APC on pol-P's dRP-lyase activity (Balusu et al.,

2007). They found that an APC wt peptide was able to block pol-P's dRP-lyase activity in a

dose-dependent manner indicating that APC blocks SP-BER by modifying pol-P's dRP-lyase

activity. Moreover, they were also able to show that the strand-displacement synthesis of LP-

BER, which is blocked by APCwt, was unaffected with the pol-P mutant (T79/K81/R83) protein.

These results indicated that the physical interaction of APC with pol-P is necessary to block its

strand-displacement synthesis. Thus, it would appear that APC is a novel accessory protein in

both SP and LP-BER.

The Interaction of APC with Fen-1 and its Implications in BER

Previous studies have shown that APC can block pol-P strand-displacement synthesis in

LP-BER and interact with Fen-1 in vitro (Jaiswal et al., 2006; Narayan et al., 2005). In the

current study these findings have been extended to examine whether APC and Fen-1 interact in

vivo, and if this interaction can block Fen-1 5'enduonuclease and 5'-3' exonuclease activities

providing another method of inhibiting LP-BER. Furthermore, critical amino acid residues on

Fen-1 and APC that participate in the Fen-1 and APC interaction have been determined. Lastly,

immunohistochemical analysis and Western blot analysis of MMS-treated cells have suggested a









novel role for APC whereby it prevents Fen-1 from entering the nucleus after exposure to DNA-

alkylating agents.

Through Y2H analysis it was determined that APCwt and Fen-1 could interact in vivo. In

light of this fact, it was decided to narrow down the interaction site on APC for Fen-1. Since the

interaction site on APC for pol-P had already been determined, it was thought that APC may

interact with Fen-1 at the same site. Using Y2H analysis of single amino acid mutants of APC, it

was found that the interaction site of APC for Fen-1 and pol-P was in the same domain. Only

amino acids Il259 or Y1262 were necessary for the interaction of APC with Fen-1. Furthermore,

the mutation of either amino acid to alanine was enough to disrupt APC's interaction with Fen-1

suggesting that the interaction of Fen-1 may be at the surface instead of in the groove of the APC

protein. Taken together, the fact that APC interacts with pol-P and Fen-1 at the same site

indicates that APC cannot interact with both proteins at the same time. This suggests that APC

may have a preference for either protein or that pol-P and Fen-1 may compete to bind with APC.

Although how the differential interaction of these proteins occurs in vivo is not clear.

Fen-1 is the maj or protein involved in LP-BER. LP-BER can occur through two

mechanisms: strand-displacement and "Hit-and-Run" synthesis. Fen-1 plays unique roles in

each. In strand-displacement, Fen-1 cleaves the flap generated by pol-P, pol-6, or pol-s mediated

strand-displacement synthesis. In hit-and-run synthesis, Fen-1 excises a nucleotide at the 3'-side

of a nick to create a gap to be filled by pol-P (Klungland and Lindahl, 1997; Prasad et al., 2001;

Qiu et al., 2001; Shibata and Nakamura, 2002) More specifically, the two mechanisms proceed

in the following manner. Once pol-P arrives at an oxidized or reduced abasic site after APE

incision, it proceeds to perform strand-displacement synthesis until Fen-1 binds and cleaves the

displaced flap leaving a nick which is sealed by DNA ligase. Alternatively, a hit and run










mechanism of LP-BER can occur in which pol-P incorporates 1-nt and the resulting flap is

removed by Fen-1 5'endonuclease activity. Then a series of sequential events occurs in which

pol-P binds to the nick left behind by Fen-1 endonuclease activity, extends the 3'-OH displacing

the dRP moiety, fills the 1-nt gap, leaves a nick, and dissociates from the DNA. Fen-1 then binds

to the nicked DNA, removes 1-nt by its 5'-3'exonuclease activity, creates a gap, and dissociates

from the DNA. This cycle continues until DNA ligase seals the nick (Liu et al., 2005).

As APC can interact with Fen-1 and has already been shown to be involved in LP-BER, it

was hypothesized that the interaction of APC with Fen-1 may cause a blockage in Fen-1 catalytic

activity, thus blocking LP-BER. Through the use of in vitro Fen-1 activity assays it was found

that an APCwt peptide could block both Fen-1 5'endonuclease and 5'-3' exonuclease cleavage

abilities in a dose-dependent manner. Thus, APC is able to block both mechanisms of LP-BER.

When APC blocks the 5'endonuclease cleavage activity of Fen-1, it effectively blocks pol-P

directed strand-displacement synthesis of LP-BER. However, when APC blocks the 5'-3'

exonuclease activity of Fen-1 it blocks hit and run synthesis of LP-BER. These results combined

with previous data show that APC can block LP-BER in three ways: 1) By blocking pol-P

directed strand-displacement synthesis through APC's interaction with pol-P; 2) By blocking

pol-P directed strand-displacement through APC's blockage of the 5' endonuclease activity of

Fen-1; and 3) By blocking hit and run synthesis of LP-BER through APC's blockage of the 5'-3'

exonuclease activity of Fen-1.

Once it was found that APC's interaction with Fen-1 caused a block in its 5'endonuclease

and 5'-3'exonuclease activities, the next logical step was to identify the amino acids of Fen-1

necessary for its interaction with APC. Protein fragments of Fen-1 were synthesized and used in

a Far-western analysis with APC. Amino acids 346-3 80 of Fen-1 were found to interact with









APC. Most of these amino acids lie within the nuclear localization signal of Fen-1 suggesting

that APC is interfering with nuclear localization of Fen-1. Indeed, two groups of amino acids

essential for nuclear localization (K3 54, R3 55, K3 56 and K365, K366, K367) lie within the

region of amino acids 346-380 (Shen et al., 2005). Furthermore, three proteins which interact

with Fen-1 have been found to interact at Fen-1's C-terminus between amino acids 3 53-3 80

(Guo et al., 2008; Sharma et al., 2005). Based on this knowledge two sets of amino acids Fen-

1(K3 54/R3 55/K3 56) and Fen-1(K3 65/K3 66/K3 67) were chosen for further studies. These amino

acids of Fen-1 are important in nuclear localization, and due to the fact that they fell between

amino acids 353-380 which have been shown to be the interaction site of Fen-1 with other

proteins. Each amino acid was mutated to alanine and the mutant proteins were synthesized and

used in a Far-western analysis with the APCwt peptide. Both mutants were shown to reduce the

interaction between APCwt peptide and Fen-1. The Fen-1(K3 65A/K3 66A/K3 67A) mutant was

able to reduce the interaction with APC to a greater extent. These results suggested that APC

does interact with the nuclear localization signal of Fen-1. More specifically, it suggests that

APC interacts with the amino acids that have been shown to be essential for nuclear localization.

To further characterize the interaction of APC with Fen-1, the synthesized Fen-1

(K365A/K366A/K367A) mutant was used in in vitro Fen-1 activity assays to determine if the

Fen-1(K3 65A/K3 66A/K3 67A) mutant possessed 5'endonuclease and 5'-3'exonuclease

activities. Surprisingly, the Fen-1(K3 65A/K3 66A/K3 67A) mutant retained both 5'endonuclease

activity and 5'-3'exonuclease activity. It was expected that the Fen- 1(K3 65A/K366A/K3 67A)

mutant protein would lose its 5'endonuclease and 5'-3'exonuclease activities due to the fact that

the interaction of APC blocks these activities. Since the Fen-1(K3 65A/K3 66A/K3 67A) mutant

retains its endonuclease and exonuclease activities, the physical interaction between Fen-1 and









APC must be necessary for Fen-1 cleavage activity to be blocked. APC may cause a

conformational change in Fen-1 upon binding that causes the active site of the endonuclease and

exonuclease activities to become misaligned causing a blockage of Fen-1 endonuclease and

exonuclease activities.

Due to the fact that the interaction of Fen-1 with APC appears to occur at the nuclear

localization signal of Fen-1, it became necessary to determine the physiological significance of

this interaction within the cell. Immunohistochemical analysis of HCT-1 16-APC(WT) cells

treated with 500 C1M MMS for 24 h showed that most cells had an increased co-localization of

APC and Fen-1 in the cytoplasm as well as a reduction in nuclear Fen-1 as compared to control

cells. This phenomenon was only seen in HCT-116-APC(WT) cells which contained wild-type

APC. HCT-116-APC(KD) cells and LoVo cells were also treated with 500 CIM MMS, but failed

to show a reduction in nuclear Fen-1 after 24 h of treatment. Since HCT-1 16-APC(KD) cells

contain stably knocked down levels of APC and LoVo cells contain truncated APC which lacks

the DRI-domain and therefore does not interact with Fen-1, these results indicate that wild-type

APC must be present for the reduction in nuclear Fen-1 to occur. Furthermore, this reduction

occurs in response to DNA-alkylation damage. To further understand how this reduction in

nuclear Fen-1 occurs after treatment with MMS for 24 h, a time course experiment was

conducted where the aforementioned cell lines were treated with MMS for 5, 15, or 24 h. Once

again the reduction in levels of nuclear Fen-1 and increased co-localization between APC and

Fen-1 were only seen in HCT-1 16-APC(WT) cells. After 15 h of treatment with 500 CIM MMS

about half of the HCT-1 16-APC(WT) cells exhibited a reduction in nuclear Fen-1 and by 24 h

most of the HCT-1 16-APC(WT) cells exhibited a loss of nuclear Fen-1. Cells treated with MMS

for 5 h did not exhibit a loss of nuclear Fen-1, but they did exhibit increased co-localization of









APC and Fen-1 in both the cytoplasm and the nucleus. In contrast these observations were not

seen in HCT-116(KD) cells or LoVo cells that received the same treatment. Furthermore, the

increased co-localization of APC and Fen-1 was not apparent in HCT-1 16-APC(WT) cells which

did not receive the MMS treatment. These results were further confirmed through Western blot

analysis. HCT-116-APC(WT) cells and HCT-116-APC(KD) cells were treated with MMS for 5,

10, 15, and 25 h and the nuclear and cytosolic fractions were collected. These fractions were then

used in Western blot analysis to determine the levels of APC and Fen-1 in the nucleus and the

cytoplasm in MMS-treated HCT-116-APC(WT) and HCT-116-APC(KD) cells. An increase in

cytoplasmic APC was seen in HCT-116-APC(WT) cells with the maximum increase being at 15

h after MMS treatment as compared to control HCT-116-APC(WT) cells. Interestingly, levels of

nuclear Fen-1 were decreased in HCT-116-APC(WT) cells in response to MMS treatment with

the maximum decrease seen at 15 h suggesting that an increase in cytoplasmic APC may lead to

a reduction in nuclear Fen-1. As expected, this phenomenon was not seen in the cytoplasmic or

nuclear fractions of MMS-treated HCT-1 16-APC(KD) cells. Levels of APC were not detected in

the cytosolic or nuclear fractions of MMS treated HCT-1 16-APC(KD) cells, while Fen-1 levels

showed no significant change in both the cytosolic and nuclear fractions. These results support

the hypothesis that APC interacts with Fen-1 at its nuclear localization signal and blocks the

translocation of Fen-1 to the nucleus. Interestingly, the highest level of nuclear APC in HCT-

116-APC(WT) cells was seen at 25 h after MMS treatment. This correlated with a modest

increase in the levels of nuclear Fen-1 providing further support for the hypothesis that APC can

interact with Fen-1 and block its catalytic activity.

Combined, these results suggest another mechanism of APC-mediated block of BER by

retaining Fen-1 in the cytoplasm in response to DNA alkylation damage. The data suggests that









APC becomes upregulated in response to MMS treatment. Over the course of treatment, APC

interacts with Fen-1 at its nuclear localization signal in the cytoplasm and prevents it from

entering the nucleus causing a loss of Fen-1 in the nucleus due to the fact that the Fen-1 protein

cannot be replenished. In summation, APC can block LP-BER through its interaction with Fen-1

via two mechanisms. First, APC can block LP-BER by binding to Fen-1 and causing a block in

both Fen-1 5'endonuclease and 5'-3'exonuclease cleavage activities. Secondly, in response to

DNA damage, APC can block LP-BER by binding to Fen-1 at its nuclear localization signal and

prevent it from replenishing nuclear Fen-1. Whether APC interacts with newly synthesized Fen-1

and whether post-translational modifications in APC and/or Fen-1 after DNA-alkylation damage

causes cytoplasmic co-localization of APC and Fen-1 is not yet known.

Conclusions and Future Implications

Mutations in the APC gene are among the earliest events in colorectal carcinogenesis. The

blockage of BER by APC may be viewed as a paradox as APC is generally viewed as a tumor

suppressor. The blockage of BER by APC could serve as a tumor suppressor if the blockage of

BER leads to apoptosis as a result of the accumulation of DNA damage. Indeed, studies have

found that treatment of human colon cancer cells and mouse embryonic fibroblast cells with

MMS enhanced the levels of APC and blocked BER, resulting in increased sensitivity and

apoptosis of cells harboring damaged DNA (Kundu et al., 2007b; Narayan et al., 2005).

Furthermore, it has been shown that HCT-116-APC(WT) cells treated with MMS exhibited

decreased colony formation in a clonogenic assay when treated with MMS compared to HCT-

1 16-APC(KD) cells treated with MMS indicating that increased levels of APC after MMS

treatment blocks BER and decreases cell growth (Kundu et al., 2007b). These results indicate

that APC plays a role as a tumor suppressor by removing cells containing damaged DNA.

Alternatively, studies have shown that cigarette smoke condensate induces APC levels in









spontaneously immortalized normal human breast epithelial cells, blocks BER, and causes

transformation of these cells (Kundu et al., 2007a). These findings point to a role of APC in

carcinogenesis.

In conclusion, the findings of the present study indicate that APC can block LP-BER by

interacting with Fen-1 through two different mechanisms: direct interaction with Fen-1 causing

blockage of its endonuclease and exonuclease activities and by binding to Fen-1 in response to

DNA damage and preventing Fen-1 from entering the nucleus to replenish nuclear Fen-1.

Furthermore, it was discovered that the linker region of pol-P is responsible for the interaction

with APC indicating that this region of pol-P may play a role in the complex regulation of the

BER process in vivo. These proteins, including APC, may be involved in specific subpathways

of BER that could be triggered by unknown signaling. Studies characterizing these subpathways

and the signaling processes that control them will serve to shed some light on the regulation of

BER in vivo and the role of APC in this regulation.

The role of APC in the blockage of LP-BER may provide clinical implications in the

future. Since the DRI-domain is located toward the N-terminal region of the APC protein, which

is spared by the MCR region, both wild-type and mutant APC proteins (containing the DRI-

domain) may block DNA damage-induced LP-BER. Therefore, if the chemopreventative agents

that produce DNA damage can increase APC levels in the target cell, then the decreased LP-BER

and increased DNA damage may induce cell death. Thus, the link between APC and DNA repair

may prove important in the development of chemotherapeutic drugs in the future.










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BIOGRAPHICAL SKETCH

Melissa Liz Armas was born in Miami, Florida, to Cuban parents. She is the only daughter

of Mayra and Eser Armas. Since an early age, she was interested in Science. Melissa attended

Southwest Miami Senior High School, where she was an honors student and was active in the

Phi Beta Chi Science club. She was also active in the Foreign Language club and the Future

Business Leaders of America.

After graduating high school, Melissa attended the University of South Florida, where

she received a B.S. in biology. She was also part of the Honors Program. Melissa was accepted

into the Ronald E. McNair Postbacculaurete Achievement Program. As a McNair Scholar,

Melissa received her first research experience. Her research proj ect was on Brain cancer

(Glioma) cells. She was able to present her research at McNair conferences and the Honors

research symposia.

After graduating from the University of South Florida, Melissa enrolled into the IDP

program at the University of Florida to pursue her doctorate. She pursued her

doctoral research on Adenomatous Polyposis Coli (APC) and its interaction with Base Excision

Repair proteins: Its role in colorectal carcinogenesis in the laboratory of Satya Narayan. Melissa

has presented her research in departmental seminars and has had two abstracts presented at

national meetings.

Melissa plans to pursue a master' s in public health. By combining the knowledge from

her doctorate and her master' s, Melissa plans to focus on Health Policy and its relation to

minority populations.





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THE INTERACTION OF ADENOMATOUS POLYPOSIS COLI (APC) AND BASE EXCISION REPAIR PROTEINS: ITS IMPLICATIONS IN COLORECTAL CARCINOGENESIS By MELISSA LIZ ARMAS 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 2008 1

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2008 Melissa Liz Armas 2

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To my parents, Eser and Mayra, for always be lieving in me; In memo ry of my Grandmother Maria, for inspiring me and teaching me to alwa ys have faith; In memory of my Grandfather Victor whose battle w ith cancer inspired me to pursue my doctorate 3

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ACKNOWLEDGMENTS I thank God for allowing me to finish this journey and bei ng with me every step of the way. I thank my parents, Eser and Mayra Ar mas, for their guidance, love, support, and encouragement. I thank my family and friends fo r their unfaltering prayers and encouragement. I thank my mentor Dr. Satya Narayan, for givi ng me the opportunity to pursue my doctoral research in his laboratory and for sharing his knowledge with me. I thank Dr. Aruna Jaiswal for his experimental guidance and assistance. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................... .............10 CHAPTER 1 INTRODUCTION ................................................................................................................ ..11 Colorectal Cancer ...................................................................................................................11 Adenomatous Polyposis Coli (APC) ......................................................................................11 The APC gene ..................................................................................................................11 The APC Protein and its Many Functions .......................................................................12 APC and -catenin regulation ..................................................................................13 APC and its role in cell adhesion .............................................................................15 APC and its role in cell migration ............................................................................16 Role of APC in chromosomal segregation and mitotic spindle assembly ...............17 Role of APC in cell cycle regulation and apoptosis .................................................18 APC and its involvement in DNA damage and repair .............................................19 DNA Damage and Repair .......................................................................................................20 DNA Damage Checkpoints .............................................................................................20 G1/S checkpoint ........................................................................................................21 S-phase checkpoint ...................................................................................................21 G2/M checkpoint ......................................................................................................22 DNA Repair .................................................................................................................... .22 Direct damage reversal .............................................................................................23 Nucleotide excision repair ........................................................................................23 Mismatch repair ........................................................................................................23 Double-strand break repair .......................................................................................24 Base excision repair .................................................................................................25 DNA Polymerase ( pol) ...................................................................................................27 PolStructure .................................................................................................................28 Roles of Pol..................................................................................................................30 Post-translational Modifications of Poland its Effect on PolFunction ...................31 Flap Endonuclease 1 (Fen-1) ..................................................................................................3 1 Fen-1 Structure and Biochemical Characteristics ...........................................................32 Residues and Stuctural Motifs of Fe n-1 Important for Fen-1 Function ..........................33 Roles of Fen-1 .................................................................................................................33 Post Translational Modifications of Fen-1 ......................................................................35 Purpose/Significance .......................................................................................................... ....36 5

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2 MATERIALS AND METHODS ...........................................................................................42 Yeast Cell Maintenance ........................................................................................................ ..42 Yeast Two-Hybrid Assays of th e Interaction of APC with Poland Fen-1 .........................42 Maintenance of Mammalian Cells ..........................................................................................43 Generation of PolDeletion Constructs ...............................................................................43 Site Directed Mutagenesis of Pol........................................................................................44 Yeast Two-Hybrid Assay of PolDeletion Constructs, PolMutants, and APC ...............44 APC Peptides ..........................................................................................................................45 Synthesis and Labeling of the Fen-1 Substrate .......................................................................45 Fen-1 Activity in vitro ............................................................................................................46 PCR of Fen-1 Protein Fragments for use in in vitro Transcription/Translation .....................46 Site-Directed Mutagenesis of Fen-1 .......................................................................................48 Synthesis of Fen-1 Protein Fragments, Fe n-1 wt, and Fen-1 Mutant Proteins Using in vitro Transcription/Translation ...........................................................................................49 Far-Western Blot Analysis .....................................................................................................49 Pull-Down of Fen-1 wild type and FenS -1 K365A/K366A/K367A fr om TNT Synthesis Reaction Mixture .................................................................................................................50 Immunohistochemistry .......................................................................................................... .50 Western Blot Analysis of Fen-1 and APC Protein Levels in the Cytoplasm and the Nucleus in Cells Treated w ith DNA-Alkylating Agents ....................................................51 3 APC INTERACTS WITH DN A POLYMERASE BETA .....................................................52 Amino acids Thr79/Lys81/Arg83 of polare Critical for its In teraction with APC ............52 Amino Acids I1259A and Y1262A, but not Q 1256A are Required for APCs Interaction with DNA Polymerase (Pol) .........................................................................................53 4 APC INTERACTS WITH FLAP ENDONUCLEASE 1 (FEN-1) .........................................59 APC and Fen-1 interact in vivo ...............................................................................................59 The Interaction Site of APC is the Same for Fen-1 and Pol...............................................59 APC Blocks the Endonuclease and E xonuclease Activities of Fen-1 ....................................60 Amino Acids K365/K366/K367 are Important fo r the Interaction of Fen-1 with APC .........61 Fen-1(K365A/K366A/K367A) Mutant Re tains Catalytic Activity ........................................63 APC Prevents the Entry of Fen-1 into th e Nucleus after Exposure to DNA-Alkylating Agents ........................................................................................................................ .........64 5 DISCUSSION .................................................................................................................. .......83 The Interaction of APC and DNA Polymerase and its Implications in BER ......................83 The Interaction of APC with Fen1 and its Implications in BER ...........................................84 Conclusions and Future Implications .....................................................................................90 6

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LIST OF REFERENCES ...............................................................................................................92 BIOGRAPHICAL SKETCH .......................................................................................................107 7

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LIST OF FIGURES Figure page 1-1 Roles of APC.. ...................................................................................................................38 1-2 Structure of the APC protein..............................................................................................39 1-3 Base excision repair ...........................................................................................................40 1-4 Structure of Fen-1 ........................................................................................................ ......41 3-1 Interaction of APC with pol-deletion constructs ............................................................55 3-2 Ribbon representation of polhighlighting the position of key mutants .........................56 3-3 Interaction of APC with polmutants.. ............................................................................57 3-4 Interaction of AP C mutants with pol..............................................................................58 4-1 Interaction of Fen-1 and APC. ...........................................................................................69 4-2 Interaction of APC mutants with Fen-1 .............................................................................70 4-3 APC blocks the 5-flap e ndonuclease activity of Fen-1 ....................................................71 4-4 APC blocks the 5-flap endonuclease activ ity of Fen-1 regardless of flap size ................72 4-5 APC blocks the 5-3 ex onuclease activity of Fen-1 .........................................................73 4-6 Protein levels of in vitro transcription/translation of wild-type and fragments of Fen1..........................................................................................................................................74 4-7 Far-western analysis of the interactio n of APCwt and APC(I-A ,Y-A) peptides and Fen-1 protein fragments.. ...................................................................................................75 4-8 Protein levels of in vitro transcription/transl ation of wild-type and mutant Fen-1. ...........76 4-9 Far-western analysis of the interaction of Fen-1 wild -type and mutant proteins ..............77 4-10 Fen-1(K365A/K366A/K367A) mutant re tains 5-flap endonuclease and 5-3 exonuclease activity ...........................................................................................................78 4-11 Imunohistochemical analysis of APC and Fen-1 co-localization in HCT-116APC(WT) and HCT-116-APC(KD) cells. .........................................................................79 4-12 A time-course analysis of co-localiza tion of APC and Fen-1 in colon cancer cells treated with MMS. ............................................................................................................. 80 8

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4-13 A time-course analysis of co-localizati on of APC and Fen-1 in LoVo cells treated with MMS. ..................................................................................................................... ....81 4-14 A time-course analysis of nuclear and cy tosolic protein levels of APC and Fen-1 in MMS-treated colon cancer cells ........................................................................................82 9

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10 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 THE INTERACTION OF APC AND BASE EXCISION REPAIR PROTEINS: ITS IMPLICATIONS IN COLORECTAL CARCINOGENESIS, By Melissa Liz Armas August 2008 Chair: Satya Narayan Major: Medical Sciences -Molecular Cell Biology Mutations in the adenomatous polyposis coli (APC) gene are among the earliest events which lead to the development of colorectal cancer. The APC protein is critical for the maintenance of the dynamics of colonic epithelial cells. APC plays a role in the regulation of catenin, cell cycle contro l, cell-cell adhesion, chromosomal stability, chromosomal segregation, and apoptosis. Previous studies ha ve shown that the APC gene is upregulated in response to DNA damage. However, the biological significan ce of the DNA damage induced APC levels is not yet known. We hypothesize that the DNA dama ge-induced APC levels may play a role in DNA repair and hence may be involved in the early stages of initiation of colorectal carcinogenesis. Interestingly, it has recently been found that APC can interact with two base excision repair (BER) proteins, DNA polymerase (pol) and Flap endonuclease 1 (Fen-1), and block pol-directed BER. In the present study, th e critical amino acid residues of poland Fen1 required for their interaction with APC have b een identified. Furthermore, two mechanisms for blockage of long patch (LP)-BER by APC through its interact ion with Fen-1 have been discovered.

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CHAPTER 1 INTRODUCTION Colorectal Cancer Colorectal cancer is the second to fourth mo st common cancer in the industrialized world. It is estimated that 148,810 new cases of colore ctal cancer will occur in 2008. Of these, 49,960 will result in death (Society, 2008American Cancer Society). Colorectal cancer is usually divided into two forms: familial or hereditary cases and sporadic cases. Of the hereditary cases there are two types which are characterized: hereditary non-polyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP). HNPCC is caused by mutations in genes encoding for DNA mismatch repair proteins. At least four genes have been shown to cause HNPCC: MSH2, MLH1, MSH6, and PMS2 (Baglioni and Genuardi, 2004). FAP arises almost exclusively from germline mutations in the adenomatous polyposis coli ( APC ) gene. Interestingly, loss of APC function is an extremely early event in the deve lopment of sporadic colorectal tumors. This suggests that APC is critical in the maintenance of the colon (Nathke, 2004). Adenomatous Polyposis Coli (APC) The APC gene encodes a large multi-domain protein which plays integral roles in many aspects of the cell. Traditi onally known as a tumor suppresso r, APC plays a role in many functions that are critical fo r cell survival. Interestingly, somatic mutations in the APC gene are found in approximately 80% of all sporadic colo rectal tumors. These mutations almost always result in a truncated APC protein product which lacks some of the critical tumor suppressor functions of APC such as -catenin regulation. The APC gene The APC gene is located on chromosome 5q21-q22, consists of 8535 nucleotides and spans 21 exons. The APC gene encodes a 310 kDa protein wh ich is composed of 2843 amino 11

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acids in its common isoform (Jai swal et al., 2004). Approximately 75 percent of the coding sequence is located on exon 15, which appears to be the most common target for both germline and somatic mutations of APC (Beroud and Soussi, 1996). Hypermethylation of the APC gene promoter has been obser ved in colorectal, melanoma, lung, oesophogeal, gastric, pancreatic, and hepatic cancers (Clement et al., 2004; Esteller et al., 2000; Grote et al., 2004; Tsuchiya et al., 2000). Methylation occurs at CpG sites in the promoter region of the APC gene and is mediated by the enzyme DNA methyl transferase 1 (DNMT1). DNMT1 induces methylation of the APC promoter by methylating the CpG region around the CCAAT-box. Once methylated, ch romatin conformational changes occur which block the binding of the transcription factor CBF to the CCAAT box, silencing APC gene expression (Deng et al., 2004). Intriguingly, it has been found that DNMT1 gene expression is regulated by APC mutations in an indirect manner (Campbe ll and Szyf, 2003). The above findings indicate that hypermethylation of the APC promoter may provide an alternative mechanism of APC gene inactivation during early stag es of carcinogenesis. The APC Protein and its Many Functions APC is a large multi-domain protein, which is present in both the cytoplasm and the nucleus. It serves many functions in the cell as demons trated by its structure (Fig. 1-2). Along the Nterminus an oligomerization domain, a homology domain, an armadillo repeat region and two nuclear export signals are present. The oligom erization domain has been shown to be a binding site for APC mutants. Studies have shown that APC mutant proteins that re tain at least the first 171 amino acids are able to bind to this site and produce a dominant negative effect (Dihlmann et al., 1999; Su et al., 1993). However, a study i nvolving the overexpressi on of a truncated APC protein in a mouse model seemed to contradict these findings when the mice did not form tumors (Smits et al., 1999). The armadillo repeat has be en designated a protein-pr otein interaction site 12

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and has been shown to bind PP2A, Asef, and KA P 3 (Jimbo et al., 2002; Kawasaki et al., 2000; Seeling et al., 1999). A number of 15-and 20-ami no acid repeats are found in the center of the protein which have been shown to bind -catenin. Nestled in between the amino acid repeats are axin binding sites, nuclear export signals, and the mutation cluster region. The mutation cluster region (MCR) is located between codons 12801513 and harbors over 60% of all somatic mutations in the APC gene (Miyoshi et al., 1992). Mutations that occur in the MCR result in a truncated protein that lacks most of the C-terminus, which is ho me to several important sites on the APC protein. It contains the basic domain, which is the site of microtubule binding, and binding sites for the proteins EB1 and hDLG (B ienz, 2002; Erdmann et al., 2000; Matsumine et al., 1996; Polakis, 1997; Su et al ., 1995). Due to its numerous inte ractions with a variety of proteins it is expected th at APC plays various roles in the cell (Figure 1-1). APC and -catenin regulation The involvement of APC in the regulation of -catenin occurs th rough several routes. APC is part of the destruction complex, which is activated in the absenc e of a Wnt signal, along with axin and glycogen synthase kinase-3(GSK3). APC causes phosphorylation and destruction of -catenin through the ubiquitin proteasome pathway (Peifer and Polakis, 2000). A recent study suggests that different domains of APC are involved in the regulation of phosphorylation and ubiquination of -catenin (Yang et al., 2 006). The destruction of -catenin keeps it from activating the T cell factor (Tcf) fa mily of transcription f actors. Targets of this family of transcription factor s include the oncogene c-myc, cy clin D1, the oncogene c-jun, and the matrix metalloproteinase matrilysin (Crawford et al., 1999; Nateri et al ., 2005; Orford et al., 1997; Shtutman et al., 1999; Tetsu and McCormic k, 1999). When APC is mutated, it prevents GSK3 -mediated phosphorylation of -catenin, sparing -catenin from ubiquitination and 13

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proteasomal degradation. The stable -catenin accumulates in the nuc leus and binds with the Tcf family of transcription factors leading to aberrant gene expressi on, which is associated with cell proliferation and cell cycle progression (Hende rson and Fagotto, 2002). Furthermore, APC has been shown to be invo lved in the export of -catenin from the nucleus, thus reducing its ability to interact with Tcf transcription factors (Henderson, 2000). APC has also been shown to sequester -catenin in the nucleus with the aid of C-terminal binding protein (CtBP), and once again, prevent its binding to Tcf transcripti on factors (Hamada and Bienz, 2004). Similarly, cytoplasmic APC has been shown to sequester cytoplasmic -catenin. When levels of cytoplasmic -catenin are excessively elevated, it induces phophorylatio n of APC by casein kinase1, GSK3 and unknown kinases. Once phosphorylated, APC is able to retain -catenin in the cytoplasm by keeping it bound in a complex preventing the entry of -catenin into the nucleus and the activa tion of Tcf signaling (Seo and J ho, 2007). Moreover, APCs role in catenin regulation suggests a role for APC in cell cycle progression. A pathway has been elucidated which shows that APC can regulate Cdk4 expression as a result of its interaction with -catenin. Through the use of an inducible APC gene in HT29 colon cancer cells, He and colleagues (1998) discovered that c-myc was a target gene of the APC pathway. They found that when APC was induced c-myc expression was repressed (H e et al., 1998), and that c-myc expression was activated through Tcf-4 which is activated by -catenin. These results lead to a proposed pathway for c-myc expression in colorectal epithe lial cells. In normal colorectal epithelial cells, wild-type APC prevents -catenin from forming a complex with Tcf-4 and activating c-myc However, in colorectal tumors which have an APC mutation or activating catenin mutation, -catenin/Tcf-4 complex formation is increased leading to overexpression of cmyc and the promotion of neoplasti c growth (He et al., 1998). 14

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Further studies proved that Cdk4 is the targ et of the aforementioned pathway (Hermeking et al., 2000). After c-myc overexpression was carried out in human umbilical vein cord cells (HUVEC) using adenovirus, a tran script encoding Cdk4 was found to be induced. This proved to be most intriguing since Cdk4 is involved in cell cycle regula tion. To further confirm these results a mutant c-myc was overexpressed in HUVEC cells to s ee if the Cdk4 transcript was still induced. As expected, Cdk4 failed to be induced in the presence of the mutant c-myc These results suggest that c-myc is involved in the direct re gulation of Cdk4 mRNA. Moreover, a concordant increase in c-myc and Cdk4 expression is found in colorectal cancers when compared to normal colon cells (Hermeking et al., 2000). Based on these data the aforementioned model with APC has been amended. In normal colorect al epithelial cells, w ild-type APC prevents catenin from forming a comple x with Tcf-4 and activating c-myc Since c-myc is not activated, Cdk4 will not be activated and this prevents cdk4 from phosphorylating and down-regulating important tumor suppressor genes such as the retinoblastoma (Rb) gene. However, in colorectal tumors which have an APC mutation or an activating -catenin mutation, -catenin/Tcf-4 complex formation is increased leading to over expression of c-myc. Over expression of c-myc leads to an overexpression of Cdk4, which can phosphorylate the Rb gene causing it to become down-regulated and promote neoplastic growth. APC and its role in cell adhesion APC is a part of the cell-cell adhesion comple x with E-cadherin, since it directly binds to -catenin and actin filaments as well as -catenin. In Drosophila mutational inactivation of APC causes delocalization of -catenin, gaps between adhesi ve plasma membranes, and a mislocalization of oocytes resulting from a fa ilure of cadherin based adhesion (Hamada and Bienz, 2002). Moreover, Hughes et al., found that enterocytes from C57BL/6J-min/+ (Min/+) 15

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mice showed a reduced associ ation between E-cadherin and -catenin in adherens junctions (Hughes et al., 2002). Min/+ mice serve as an an imal model for familial adenomatous polyposis. These mice have one APC allele which carri es a truncation mutation at codon 850 and one normal APC allele (Su et al., 1992). Furthermore, Faux and colleagues were able to show that restoration of full length APC into SW480 cancer cel ls, which contain truncated APC, resulted in enhanced cell adhesion (Faux et al., 2004). The e nhanced cell adhesion was attributed to the involvement of APC in post-translational regula tion of E-cadherin localization. When full length APC was restored E-cadherin lo calization changed from the cytoplasm to the cell membrane resulting in tighter adherens junctions (Faux et al., 2004). APC and its role in cell migration Wild-type APC is necessary for the upward movement of colonic epit helial cells along the crypt villus axis. During migration colonic epith elial cells are differentiated into absorptive, secretory, paneth, and endocrine cells. Mutation or loss of APC causes aberrant migration of these cells and the formation of polyps as well as altered differentiation in colonic epithelial cells (Moss et al., 1996; Sansom et al., 2004). Kroboth and colleagues showed that loss of APC results in immediate defects in cell migration in cells that constitutively lack APC and in cells where APC loss was induced conditionally using a CreFlox system (Kroboth et al., 2007). Migration was affected in both cell monolayers and in single cell movements. Furthermore, APC can regulate cell polarity and migration through th e actin cytoskeleton. APC can interact with a Rac-sp ecific guanine nucleot ide exchange factor (Asef1) through its armadillo repeats. Once activated, Asef activates Rac 1, leading to increased cell spreading, membrane ruffling and lamellipodia formation (Han son and Miller, 2005; Kawasaki et al., 2000). Additionally, it has been shown that truncat ed APC constitutively activates Asef. The 16

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constitutively active Asef causes a decrease in E-cadherin mediated cell-cell adhesion and promotes cell migration (Kawasaki et al., 2003). A recent study has shown that APC can also interact with Asef2, a relative of Asef1. Through this interaction it was found that APC activates Cdc42, which is involved in actin polymeriza tion and cell polarity (Kay and Hunter, 2001). Moreover, it has been found that IQ-motif containing GTPase act ivation protein 1 (IQGAP1), a scaffold protein involved in the control of actin filaments and microtubules as well as an effector of Rac 1 and Cdc42, recruits APC to specific site s in migrating cells. A complex is then formed between IQGAP1, APC, Rac 1, Cdc42, and the mi crotubule stabilizing protein CLIP-170. If either APC or IQGAP1 is removed, polarized mi gration as well as formation of the actin meshwork is inhibited (Aoki and Taketo, 2007; Briggs and Sacks, 2003; Watanabe et al., 2004). Role of APC in chromosomal segregation and mitotic spindle assembly The ability of APC to interact with Bub kinase s and microtubules suggests a role for APC in chromosomal segregation and mitosis. APC has been found at the ends of mictrotubules in a complex with the checkpoint proteins Bub1 and Bub3, suggesting a role of APC in mitosis. It has been shown that APC localizes to spindle poles, spindle microtubules, and kinetochores (Dikovskaya et al., 2004; Fodde et al., 2001; Kapl an et al., 2001; Louie et al., 2004) and the loss of APC or expression of APC truncation mutants, similar to those found in cancer cells, leads to defects in the mitotic spindle. Moreover, the sp indle checkpoint is compromised allowing cells to progress through mitosis with inco mplete chromosomal alignment or attachment (Dikovskaya et al., 2004; Draviam et al., 2006; Green and Kapla n, 2003; Green et al., 20 05; Tighe et al., 2004). Incomplete chromosomal alignment or attachme nt can lead to aneuploidy and tetraploidy causing chromosomal instability. Along with its roles in chromosomal segregati on and mitosis, APC has also been found to bind the ends of microtubules a nd stabilize them. It is thought that stabili zation of microtubules 17

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by APC occurs through its ability to interact with EB1 (Morrison et al., 1998; Nakamura et al., 2001; Su et al., 1995). APC may form a complex with EB1 and the formin family protein, mDia, in order to regulate the microtubule network an d its stabilization (Wen et al., 2004). Further evidence is seen in the fact that APC has been found to interact with the microtubule destabilizing protein kine sin (KIF2C). Moreover, lo ss of APC causes microtubule depolymerization to occur much faster than is seen in cells with wild-type APC. Compromised microtubule stability can lead to failures in mitotic spindle assembly and regulation of the microtubule network (K roboth et al., 2007). Role of APC in cell cycle regulation and apoptosis Overexpression of APC has been shown to block cell cycle progression from G0/G1 to S phase by negatively affecting cyclin-cdk complexes. Increased APC has been found to arrest the cell cycle in the G0/G1-S phase and G2/M phase (Baeg et al., 1995; Hein en et al., 2002). It is thought that the APC binding partner, human discs la rge (hDLG) forms a complex with APC that regulates the G1/S phase transition. APC which lacks th e hDLG binding site does not block cell cycle activity as strongly suggesting that the interaction with hDLG is necessary to downregulate cell growth (Ishidate et al., 2000). However, the role of APC in cell cycle control with damaged DNA is not yet fully understood. The role of APC in apoptosis is controversial. The precise mechanism is not yet clear. In the colon, increased levels of APC can induce cell death under certain circumstances. Studies have been conducted using the HT29 colon cancer cell line which show that expression of full length APC in this cell line leads to a decrease in cell growth and apoptosis (Morin et al., 1996). Additional studies with this cell line have shown that expression of full length APC downregulated the anti-apoptotic factor Survivin (Zhang et al., 2001). In addition, studies have shown that APC induces apoptosis by accelerating apoptosis-associated caspase activities (Chen et al., 18

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2003; Steigerwald et al ., 2005). Furthermore, Qian and coll eagues have shown that caspase cleavage of APC and release of its amino terminal domain may be required for APC to accelerate apoptosis-associated caspase activ ities independent of transcript ion. Lastly, some studies have suggested that a decrease in APC levels is associated with apopt osis. It is possible that both increased and decreased levels of APC play a role in apoptosis and that different factors such as cellular signals play a ro le in whether increased or decreas ed levels of APC are involved in apoptosis (Brancolini et al., 1998; Browne et al., 1998; Browne et al., 1994; Jaiswal et al., 2002; Jaiswal and Narayan, 2004; Schmeiser and Grand, 1999; Williams et al., 1996). APC and its involvement in DNA damage and repair The basal level expression of APC occurs th rough Upstream Stimul ating Factors 1 and 2 (USF-1 and USF-2) (Narayan and Jaiswal, 1997). However, APC gene expression is inducible and upregulated by p53 in response to DNA dama ge (Jaiswal and Narayan, 2001; Narayan and Jaiswal, 1997). The presence of damaged DNA usually leads to one of thr ee possibilities. It can cause the cell to undergo DNA repair, apoptosis, or if the DNA damage persists and the cell survives it can lead to cell transformation. Since APC gene expression is upr egulated in response to DNA damage, it is possible that APC is playi ng a role in one or al l of these scenarios. However, it was decided to focus on the involvem ent of APC in DNA repair since a defect in DNA repair is one of the initial steps in car cinogenesis. Recently, a DNA Repair Inhibitory (DRI) domain was identified in APC that allo wed it to bind with two base excision repair proteins DNA polymerase (pol) and Flap endonuclease-1 (Fen-1 ). Through this interaction it appeared that APC was able to block poldeoxyribose phosphate (dRP) lyase activity,shortpatch base excision re pair (SP-BER), pol-mediated long-patch base excision repair (LP-BER), and Fen-1 cleavage activity (Balusu et al., 2007; Ja iswal et al., 2006; Narayan et al., 2005). In 19

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addition to the DRI-domain, the APC protein al so contains a mutati on cluster region (MCR). Approximately, 60 percent of the APC mutations in colon cancer arise from the MCR. Mutations in this region lead to a more severe polyposis than patients with mutations outside the MCR (Narayan and Roy, 2003). It is of interest to note that the DR I-domain is not in this region, indicating that both wild-type and truncated APC retain the ability to interact with these proteins and block BER. DNA Damage and Repair The genomes of eukaryotic cells are under constant attack by endogenous and exogenous sources. Exogenous exposure arises from environmental agents such as ultraviolet (UV) radiation, ionizing radiation (IR), and various genotoxic ag ents while endogenous exposure arises as by-products of cellular metabolism a nd includes hydrolysis, oxidation, alkylation, and mismatch of DNA bases (Houtgraaf et al., 2006). Damaged DNA that is not repaired can lead to genomic instability, apoptosis, or mutagenesis which can eventually lead to carcinogenesis. Fortunately, the cell has devel oped various DNA damage checkpoi nts and DNA repair pathways to deal with the genomic insults it encounters. DNA Damage Checkpoints DNA damage triggers the activ ation of cell cycle checkpoints. These checkpoints are activated during the differe nt phases of the cell cycle. The eu karyotic cell cycle contains the following phases, gap (G)1, synthesis (S), gap (G) 2, mitosis (M) and one phase outside of the cell cycle G0 (Kastan and Bartek, 2004; Nyberg et al., 2002). During G1 the cell becomes larger and starts synthesizing RNA and proteins which are needed for transcription and translation. The synthesis of RNA and pr otein continues through G2. In S phase, the cell replicates its DNA so that one copy can be passed on to each daughter cell. During G2 the cell continues to grow and makes extra proteins to ensure that two viable cells can be formed. Lastly, the cell will enter M 20

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phase where the chromosomes will be aligned an d two genetically identi cal daughter cells will be formed. As long as cells are dividing, the cycl e will continue. When cells stop dividing they are said to be in G0. DNA damage checkpoints generally occur during the transition of each phase. They cause cell cycle arrest and allow the cells DNA repa ir machinery to repair the damaged DNA. However one checkpoint occurs during a cell cycle phase. The checkpoints are the G1/S checkpoint, the intra-S checkpoint, and the G2/M checkpoint (Ishikawa et al., 2006). G1/S checkpoint In response to DNA damage the G1/S checkpoint serves to prevent the transition between the G1 phase and the S phase in the cell cycle. Once DNA damage is sensed a myriad of signaling cascades serve to cause G1 arrest. Briefly, ATM (ataxiatelangiectasia) or ATR (ATM and RAD related) which are both known as DNA damage sensors, directly phosphorylate the p53 in its transactivation domain on Ser 15. More over, MDM2 is also targeted by ATM/ATR and CHK2/CHK1 kinases due to its involveme nt in p53 regulation. Activation of p53 and MDM2 allows the regulation of the transcri ption of target genes p21CIP1/WAF1. Once activated, p21CIP1/WA F1 inhibits the G1/S cyclin/cdk promoting complexes (Wahl and Carr, 2001). Specifically, the p21CIP1/WA F1 signal inhibits the RB/E2F pathway and thus prevents G1/S promotion. S-phase checkpoint During the S-phase of the cell cycle DNA replication occurs. In addition to DNA damage, S-phase checkpoints can be triggered as a result of difficulties with DNA replication such as stalled replication forks. There are thr ee types of S-phase checkpo ints: 1) the replication dependent checkpoint, 2) the replication indepe ndent checkpoint, and 3) the S-M checkpoint. The replication dependent checkpoint and the S-M checkpoint are dependent upon DNA replication while the re plication independent checkpoint is triggered by double strand breaks. All 21

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three of these checkpoints share components (Barte k et al., 2004). Key components in all three of the S-phase checkpoints include, RAD17, and the RAD9-RAD1-HUS1 complex, replication protein A, ATR, the ATR interacting protein (ATRIP), the ATR activator Top BP1, and the mediator protein CLASPIN. In response to DNA damage all three checkpoints have a similar response. Once DNA damage is detected, the RAD9-RAD1-HUS1 complex is loaded onto the area surrounding the DNA lesion by RAD17. This act ion allows ATR-medi ated phosphorylation and activation of CHK1 kinase, causing cell cycl e arrest (Mordes et al ., 2008; Zou et al., 2002). G2/M checkpoint The main function of the G2/M checkpoint is to preven t cells with unrepaired DNA damage from entering mitosis. Cells are prev ented from entering mitosis in CHK1 kinasemediated manner. Once activated, the G2/M checkpoint begins with ATM/ATR-mediated phosphorylation of CHK1/CHK2 kinases and p38 kinase, causing degrad ation, inhibition, or sequestration of cdc25 phosphatases. Once i nhibited, the cdc25 phos phatases can no longer activate CDK1 which allows the cell to proceed into mitosis. Thus, the cell cycle is arrested (Nyberg et al, 2002; (Kas tan and Bartek, 2004). DNA Repair If left unrepaired, DNA damage interferes with replication and tran scription leading to apoptosis. Furthermore, continuous DNA damage can cause accumulation of genetic mutations that lead to carcinogenesis. Howe ver, the cellular system is equipped with repair mechanisms to deal with damaged DNA. There are six main DNA repair mechanisms or pathways: DNA damage reversal, nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination repair (HR), non-homologous end-join ing (NHEJ) repair, and base excision repair (BER). The type of DNA damage present determines the pathway of DNA repair that is used. 22

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Direct damage reversal In direct damage reversal, the damage to th e base is repaired by methyltransferases or oxidative demethylases. In general, O-alkylations are repair ed by alkyltransferases and Nalkylations tend to be repaired by oxidative demethylases. These enzymes work by transferring the alkyl or methyl group from the damaged base onto the enzyme. This action auto-inactivates the enzyme. Thus, the enzyme is limited by the amount available in the area of the damaged base (D'Incalci et al., 1988; Gerson, 2004; Middleton and Margison, 2003; Sabharwal and Middleton, 2006). Nucleotide excision repair DNA adducts that form large bulky lesions and covalent linkages between adjacent pyrimidines resulting from UV exposure are repa ired through nucleotide ex cision repair (NER). NER has two direct pathways termed transcription-coupled repair (TCR) and global genomic repair (GGR). TCR serves as a system that detects and removes DNA damage that blocks the progression of RNA polymerase II (Hanawalt, 2002). GGR is slower than TCR and serves as a system that surveys the entire genome for da mage. Both pathways follow a similar process. Briefly, DNA damage is recognized by the XPCHR23B complex in GGR and RNA polymerase II in TCR and the damage is verified by XP A. Subsequently the DNA is unwound by XPB and XPD helicases that are in a comp lex with TFIIH basal transcrip tion initiation factor. The DNA is then incised by XPF and XPG on the damaged st rand. Lastly, the damaged portion of the DNA is removed and the repair is completed by a DNA polymerase followed by DNA ligase. Two additional proteins are required for TCR, CSB and CSA (Hanawalt, 2002). Mismatch repair The mismatch repair (MMR) system removes base-base mismatches and insertion and deletion loops that are formed during DNA replica tion (Jiricny, 2006). It is also important in the 23

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maintenance of repeated sequences. Mutations in the MMR system are associated with a predisposition to cancer (Ben Yehuda et al., 2000; Karran, 1996; Skinner and Turker, 2005). Briefly, base mispairs and small insertion or deletions are recognized and the recognized lesions are excised. Lastly, the DNA is resynthesized an d repair is complete (Neri et al., 2005). Double-strand break repair Double strand breaks are repaired by the ce lls homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair systems. Repair of these lesions is important because these lesions can lead to genomic instability, apoptosis and/or mutations due to deletions, insertions, translocations, and an euploidy. HR is an error-free re pair pathway. In this pathway homologous sequences are aligne d on the sister chromatid (Hefferin and Tomkinson, 2005; Khanna and Jackson, 2001; Scott and Pandita, 2006) The sister chromatid serves as a template so that the correct information can be copied to the missing information on the locus where the break occurred. This process is mediated by RAD 5, is aided by proteins in the RAD52 epistasis group (Helleday, 2003; Jackson, 2002), and usually occurs during the G2 phase of the cell cycle (Hefferin and Tomkinson, 2005; Khanna and Ja ckson, 2001; Scott and Pandita, 2006). On the other hand NHEJ is an error prone pro cess that usually occurs during the Go/G1 phase of the cell cycle. In NHEJ, the two broken ends are fused together irrespective of homology. The process begins with Ku70/Ku80 heterodimer binding to the broken ends of the DNA. Then the broken ends are processed for ligation with the aid of the Artemis-DNA-PKCs complex which is recruited by the Ku heterodimer. Next, gap f illing by a DNA polymerase of the pol-X family occurs and the resulting nick s are covalently jo ined by the XRCC4-DNA ligase IV complex (Lieber, 1999; Lieber et al., 2003). Due to the fact that this process disregards homology, NHEJ can lead to insertions or deletions of filler DNA making it error prone (Gorbunova and Levy, 1997; Sargent et al., 1997; Seluanov et al., 2004) 24

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Base excision repair Base excision repair (BER) is a pathway that removes damaged DNA, specifically abasic DNA (Fig. 1-3). It is composed of two sub-path ways single nucleotide base excision repair which is also referred to as short-patch (SP)-B ER and multi-nucleotide base excision repair which is also referred to as long-patch (LP)-BER. The pathway used depends on the size of the repair gap and the type of DNA damage. For ex ample, if the damaged DNA base is reduced or oxidized it would go through LP-B ER (Klungland and Lindahl, 1997; Podlutsky et al., 2001). In both SP-BER and LP-BER the process begins with the removal of the damaged base. A DNA glycosylase recognizes the damaged base and cleaves th e glycosidic bond between the sugar and the base leaving an apurinic/apryrimidinic (AP) or abasic site. AP-endonuclease 1 (APE-1) then comes in and makes an incision at the 5end of the AP-site of the DNA backbone. The decision is then made whether to use th e SP-BER or LP-BER depending on the type of DNA damage. During SP-BER, the deoxyribose phosphate (dRP) lyase activity of DNA polymerase (pol), which is located in the 8 kDa domain of pol, removes the 5dRP left behind by APE-1 resulting in a 5 phosphorylated nicked DNA strand (Matsumoto and Kim, 1995). Polthen uses its polymerase activity, which is located in its 31 kDa C-terminal domain, to synthesize the correct base at the site of the damaged base. The remaining nick is sealed by DNA ligase I (Mitra et al., 2001). When the AP-site is oxidized or reduced it becomes chemically resistant to the dRP lyase activity of poland must be repaired through LP-BER. In this case, polcan synthesize up to 13 nucleotides creating a 5 overh ang or flap that must be removed to complete LP-BER. The flap is removed by flap endonuclease-1 (Fen-1) leaving behind a nick which is sealed by DNA ligase I (Lieber, 1997; Podlutsky et al., 2001). In addition to pol, poland polcan also 25

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participate as the main polymerase that synthe sizes the 13 nucleotides in strand displacement synthesis (Fig. 1-3). Alternate BER pathways. Recently, a different class of sub-pathways of BER have been discovered. The common denominator in all these sub-pathways is a single strand break. These pathways have also been termed alternate BE R pathways (Almeida and Sobol, 2007). The main difference in these pathways is in the initiation of BER. The s ub-pathways that have been found are: nucleotide incision repair NEIL2 DNA glycosylase initiate d repair, radiation-induced DNA damage or single strand break repair (SSBR), and strand breaks generated by topoisomerase poisons. In response to the type of damage th at causes the single strand break, one of the subpathways may be initiated (Almeida and Sobol, 2007). Nucleotide incision repair occurs when APE1 acts directly on the damaged DNA bypassing the glycosylase step. It makes an incision upstream of the oxidatively damaged base leaving a 3OH and 5 phosphate cont aining the damaged base at the en ds. It is thought that this specifically happens in response to a speci fic type of oxidative damage, 5-hydroxy-2deoxycytidine (5OH-C). The resul ting product is a substrate for LP-BER and APE1 goes on to stimulate Fen-1 activity as well as DNA ligas e I activity (Dianova et al., 2001; Ischenko and Saparbaev, 2002; Ranalli et al., 2002). NEIL2 DNA glycosylase-initia ted repair occurs through th e use of the bi-functional DNA glycosylases NEIL1/NEIL2. In addition to re moving the oxidized base similarly to monofunctional glycosylases such as OGG1 and NTH1, NEIL1 and NEIL2 further process the AP site to yield a 3 phosphate and 5phosphate at the margins. Due to the fact that APE 1s 3 phosphatase activity is not very strong, this pa thway utilizes another enzyme to process the resulting NEIL1/NEIL2 product. Instead, polynucl eotide kinase phosphatase (PNKP) is required 26

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to remove the 3 phosphate. A complex then forms between NEIL1/NEIL2, pol, XRCC1, DNA ligase III, and indirectly with PNKP. Repair then proceeds in a manner similar to SP-BER (Caldecott, 2002; Wieder hold et al., 2004). Radiation-induced DNA damage causes single stranded breaks in the DNA. The pathway begins with PARP-1 recognizing and binding to single strand breaks. XRCC1 is then recruited and repair proceeds in the trad itional manner of SP-BER. On the other hand, strand breaks generated by topoisomerase poisons are repaired in a different manner. The lesion in this case begins as a DNA:topoisomerase 1 (Top1) comp lex creating a toxic single strand break. The enzyme tyrosyl DNA phosphodiesterase (Tdp 1) cat alyzes the hydrolysis of Top1 from the DNA leaving a gap with a 3 phosphate and a 5 hydroxyl. PNKP can then transfer the 3 phosphate to the 5 end. At this point, ga p filling can proceed through poland it is thought that XRCC1 also participates (Plo et al., 2003). APCs proposed role in BER. As previously mentioned it was found that APC was able to interact with polin vivo and that APC was able to block pol-directed strand-displacement synthesis during LP-BER (Naray an et al., 2005). Furthermore, in 2006, Jaiswal and colleagues found that APC also interacted with Fen-1 in vivo and in vitro (Jaiswal et al., 2006). Combined, these findings indicate that APC is playing an impor tant role in modulating BER activity. Specifically, these data suggest th at APC is playing a role in LP-BER. DNA Polymerase ( pol) DNA polymerase is one of the smallest eukaryotic DNA polymerases known to date. It is one of several known mammalian cellular DNA polym erases. Its major role is in DNA repair, specifically, BER. It has been s hown to participate in both SP-BER and LP-BER (Narayan et al., 27

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2005). Furthermore, it is embryoni c lethal indicating that polis necessary for survival (Niimi et al., 2005). PolStructure Polis a 39 kDa protein composed of 335 ami no acids (Prasad et al., 1998b). It contains two domains and one protease sens itive hinge region. The first domain is the 8 kDa lyase domain and the second domain is the 31 kDa polymerase do main. The 8 kDa lyase domain is located at the amino terminus and contains the lyase activ ity that is responsible for removing the 5deoxyribose phosphate that is generated after incision of the damaged DNA by APE1 in SPBER. The active site of the lyase domain has been identified as Lys72 (Prasad et al., 1998a). Moreover the residues that form the active site pocket have been identified. The active site pocket is defined by the following residues: Glu26, Ser30, His34, Lys35, Tyr39, Lys68, Lys72 (Batra et al., 2005; Prasad et al., 2005). The lyase r eaction proceeds through a -elimination reaction with the active site pock et residues participating in the lyase reaction itself. Tyr39 is proposed to perform nucleophilic attack. Lys35 is proposed to be involved in enzyme mediated hydrolysis while Glu 26 and Ser 30 are proposed to participate in proton ab straction. However, the reaction is not well underst ood (Prasad et al., 2005). In addition to lyase activity, the 8 kDa lyase domain contains single stranded DNA bind ing activity (Prasad et al., 1993). The 8kDa lyase domain is alpha-helical in nature and forms one of two helix-hairpin-h elix (HhH) structural motifs present in pollocated in residues 55-79 of the lyas e domain (Krahn et al., 2004; Mullen and Wilson, 1997; Pelletier et al., 1996). It has been observed to aid in the stabilization of the 90o bend in nicked DNA and/or in the stabilization of the incised DNA strand by facilitating the proper positioning of the two free DNA ends (Mat sumine et al., 1996; Sawaya et al., 1997). 28

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Like other DNA polymerases, polcontains a two metal ion cat alytic site found in the 31 kDa polymerase domain which is responsible for the catalysis of the nuc leotidyl transferase reaction (Pelletier et al., 1994; Sa waya et al., 1997). It is loca ted at the C terminus of pol. The 31 kDa domain can be broken down into three su b-domains: 1) the D-sub-domain (6 kDa) which is responsible for dsDNA binding; 2) the C-su b-domain (10 kDa) which houses the catalytic activity needed for the nucleotidyl transferase re action; 3) the N-sub-domain (12 kDa) which is involved in dNTP selection and nascent base pair binding (Beard et al., 2002). The Dand Nsub-domains are situated on opposite sides of the C-sub-domains (Sobol and Wilson, 2001). The D-sub-domain contains the other HhH structural motif in pol. It is located at residues 92-118 (Beard et al., 2002; Krahn et al., 2004; Mullen and Wilson, 1997; Pelletier et al., 1996; Sobol and Wilson, 2001). The HhH motif serves the same function as the aforementioned HhH motif located in the 8 kDa lyase domain. The two HhH mo tifs work together to facilitate the proper positioning of the two free DNA ends of the DN A substrate. The C-sub-domain possesses critical amino acids that are important for the catalytic activity of the 31 kDa domain. Residues Asp190, Asp192, and Asp256 have been shown to coor dinate the two divalent metal cations, Mg2+ that assist in the nucleotidyl transferase activity and they are known as active site residues (Date et al., 1991; Menge et al ., 1995; Sobol et al., 2000). Tw o other residues Ser180 and Arg183 have also been found to play a significant role in catalysis (Date et al., 1990; Kraynov et al., 2000). The N-sub-domain contains residues involved in polfidelity. Residues Tyr265 and Ile260 have been shown to be important in maintaining the fidelity of pol. It has been observed that the N-sub-domain closes around the nascen t base pair once the co rrect nucleotide binds (Clairmont et al., 1999; Opresko et al., 2000; Opresko et al., 1998; Shah et al., 2001; Shah et al., 2003; Starcevic et al., 2005). Polis thought to have an induced-f it mechanism for fidelity. In 29

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the induced fit model of fidelity after initial binding of the co rrect dNTP, the ternary complex undergoes conformational changes which allow cat alytic residues to be aligned for proper activation of the catalytic site. However, if the incorrect dNTP is incorporated, it is a poor fit and the alignment of the catalytic residues does not occu r correctly which causes sub-par activation of the catalytic site (Johnson, 1993; Showalter and Tsai, 2002). Additional amino acid residues shown to be involved in polfidelity are found in a loop under the C-sub-domain. Mutation of Asp246, Glu249, and Arg253 leads to a decrease in polymerase fidelity (Dalal et al., 2004; Hamid and Eckert, 2005; Kosa and Sweasy, 1999). Lastly, polcontains a protease sensitive hinge region which connects the 8 kDa lyase do main and the 31 kDa polymerase domain (Prasad et al. 1998). This region is sens itive to proteases and allows se paration of the 8 kDa and 31 kDa domains when it is exposed to proteases. Roles of PolPolplays a dual role in BER. In SP-BER, poluses its lyase activity to remove the 5deoxyribose phosphate (dRP) left behind after AP E1 incision (Matsumoto and Kim, 1995). It then uses its polymerase activity to fill the 1-nt gap left behind with the correct corresponding nucleotide. While in LP-BER, poluses its polymerase activity to perform strand-displacement synthesis. Moreover, in studies performed using poldeficient cells or in the presence of a neutralizing anti body, lack of polcaused a reduction in DNA repair capacity suggesting a role for polin LP-BER (Dianov et al., 1999). Polhas been reported to be involved in meiosis and double stranded break repair. Specifically, polhas been shown to be associated in the meiotic events of synapsis and recombination (Plug et al., 1997). It has also been shown to be involved in non-homologous end joining in yeast, where Wilson and colleagues discovered a polyeast homologue-dependent 30

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pathway for non-homologous end joining, sugges ting that a similar pathway may exist in mammals although none have been found to date (Wilson and Lieber, 1999). Post-translational Modifications of Poland its Effect on PolFunction Post-translational modifications are known to a ffect the activity of the target protein. It stands to reason that post-transl ational modifications serve to m odulate the desired activity of a target protein. In the case of polit would seem that post-translat ional modification would play a large role in affecting pols activity with regard to BER. Studies have now confirmed this thought. Polhas been found to undergo acetylation as well as methylation. In 2002, Hasan and colleagues found that polwas able to form a complex with the transcriptional co-activator p300, and that p300 was able to acetylate polin vitro and in vivo (Hasan et al., 2002). Interestingly the target for ace tylaton by p300 was Lys 72 of pol. Lys 72 is the active site for pollyase activity. Not surprisingly, this acetylation caused a block to pols lyase activity and it was not able to function properly in a reconstituted BER assay. More recently, El-Andaloussi and colleagues discovered that Arg 83 and Arg 152 of polare targets for methylation by protein arginine methyltransferase (PRMT6) in vitro and in vivo (El-Andaloussi et al., 2007; El -Andaloussi et al., 2006). This methylation stimulates pols activity. Specifically, it ca uses an increase in pols polymerase activity but not the lyase activity. Thus, the methylation of polplays a role in increasi ng the activity of BER. Flap Endonuclease 1 (Fen-1) Fen-1 is a structure specific nuc lease that has many roles. It has been found to be involved in maintaining genome stability through its involve ment in DNA replication and repair. It is embryonic lethal indicating that Fen-1 is critical for survival of the organism. Moreover, mutations in Fen-1 have been im plicated in various diseases including Huntingtons disease, 31

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myotonic dystrophy, and cancer (Zheng et al., 2007 [53]). It is thought that Fen-1 may be a novel tumor suppressor (Liu et al., 2004; Shen et al., 2005; Henneke et al., 2003). Fen-1 Structure and Biochemical Characteristics Fen-1 is a 43 kDa protein that houses th ree domains: The N-domain, I-domain, and Cdomain. The N-and I-domains are important for catalytic activity a nd are highly conserved among Fen-1 homologues (Zheng et al., 2002). The C-domain houses the proliferating cell nuclear antigen (PCNA) motif and the nucle ar localization signal and is only found among higher organisms (Frank et al., 2001; Qiu et al., 2001; Shen et al., 1998). The nuclear localization signal is critical fo r eukaryotic organisms because Fen-1 must be in the nucleus to perform its various roles. Three individual biochemical activities have been attributed to Fen-1. Harrington and colleagues discovered the 5 flap endonuclease ac tivity and the 5 to 3 exonuclease activity of Fen-1 (Harrington and Lieber, 1994). Both of these activities are housed in the same catalytic domain of the protein. Fen-1 efficiently cleaves DNA or RNA fl aps using its 5 endonuclease activity and this activity is essential for its role in DNA replication where it is involved in Okazaki fragment maturation (Bambara et al ., 1997; Lieber, 1997; Liu et al., 2004). The exonuclease activity of Fen-1 can cleave nicks, gaps, and 5end recessed double stranded DNA. It can also cleave blunt-ended DNA, albeit less efficiently (Li ndahl, 1971; Murante et al., 1994). The role of Fen-1s exonucleas e activity is not as well define d, but current thought suggests a role in Okazaki fragment maturation and apoptosis (Liu et al., 2004; Parrish et al., 2003). The most recent biochemical activity attributed to Fen-1 is a gap endonuclease (GEN) activity. Human Fen-1 was found to cleave the temp late strand of a gapped DNA fork and bubble substrates which simulate those substrates found in a stalled replication fork (Zheng et al., 2005). 32

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The cleavage was able to occur without the pres ence of a single stranded 5 DNA flap indicating that GEN activity and endonucleas e activities are separate. Residues and Stuctural Motifs of Fe n-1 Important for Fen-1 Function Several residues and structural motifs have b een identified that are important for the function of Fen-1 (Fig. 1-4). Amino acids D86, E158, E160, D179, D181 and D233 have been reported to be critical for the catalytic activity of Fen-1. Mutations of th ese residues caused a complete loss of Fen-1 endonuclease activity. Th e aforementioned residues are thought to be involved in the coordination of the two Mg2+ or Mn2+ ions to create the nuclease active site of the protein (Shen et al., 1997; Zhe ng et al., 2002). In addition, two more amino acid residues were identified (K93 and R100) that played a crucial role in catalysis. Mutation of these amino acids caused a loss of flap endonuclease activity (Qiu et al., 2004). It is thought that these amino acids may participate in the catalytic reaction itself possibly through activati on of a water molecule that participates in nucleophilic attack. Fen-1 also contains a H3TH motif which is proposed to mediate th e binding of ssDNA and dsDNA portions of the flap substrate (Hosfiel d et al., 1998). Other important amino acids involved in Fen-1 function incl ude: 1) L340, D341, F343, and F344 which are important for the interaction of PCNA with Fen-1, and 2) K354, R355, K356, K365, K366, and K367 which are critical for the nuclear localization of Fen-1 in eukaryotic cells (Frank et al., 2001; Qiu et al., 2001). Roles of Fen-1 Fen-1 has roles in DNA replication, DNA repa ir, apoptosis, and in maintaining genome stability. In DNA replication Fen-1 s main role is in Okazaki fragment maturation. In eukaryotic organisms, DNA synthesis is initiated with short RNA primers that must ultimately be removed. When the DNA replication protei ns encounter a down stream Okazaki fragment, some of the 33

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RNA primer is displaced forming a 5 flap, th at must be removed for DNA replication to continue (Arezi and Kuchta, 2000). The basic thought is that Fen1 is required to remove or degrade the RNA primers. Three models have been proposed for Okazaki fragment maturation and Fen-1 is a major player in all three models. In the first scenario, Fen-1 cooperates with RNase H to remove the RNA primer leaving a ni ck which is sealed by DNA ligase I (Turchi et al., 1994). However, it has been found that RNas e H is not required for removal of the RNA primer (Qiu et al., 1999). Thus, a Fen-1 only pa thway has been proposed. In this case, Fen-1 alone removes the entire RNA primer (Murante et al., 1995; Siegal et al., 1992; Turchi and Bambara, 1993). Lastly, a pathway involving an other endonuclease, Dna2p, has been proposed. In this pathway Dna2p physically interacts w ith Fen-1 and removes the DNA portion of the RNA primer flap. Dna2ps activity is stimulated by replication protein A (RPA). However, RPA inhibits Fen-1 activity. Once cleavage by Dna2p is complete, RPA leaves, and a shorter flap remains that is cleaved by Fen-1 (Kang et al., 2000). Fen-1s role in DNA repair ha s been discussed in the prev ious section on BER. Briefly, Fen-1 is required in LP-BER for removal of the flap formed from strand-displacement synthesis. Moreover, Fen-1 activity in LP-BER is regulated through its interaction with other BER proteins such as poland PCNA (Gary et al., 1999; Prasad et al., 2000). Additionally, Fen-1 is involved in another mechanism of LP-BER termed the Hit-and-Run mech anism. In this mechanism, polrecognizes and binds to the nicked DNA gene rated by Fen-1 as a result of the removal of the flapped DNA. Then polextends the 3OH displacing the dR P-moiety and fills the 1-nt gap leaving a nick. Poldissociates from the DNA and Fen-1 binds to the nicked DNA and removes 1-nt using its 5-3 exonuclease activity. The removal of 1-nt crea tes a gap and Fen-1 dissociates 34

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from the DNA. This cycle of LP-BER is repeat ed several times until DNA ligase seals the nick (Liu et al., 2005). Interestingly, another role for Fen-1 in DNA repair has been discovered in yeast. Yeast Fen-1 was found to interact with Pol4 and Dn l4/Lif1, which are a part of the non-homologous end joining (NHEJ) double stranded break DNA repa ir pathway (Tseng and Tomkinson, 2004). Mutations in Fen-1 have been found to increase genomic instability by causing chromosome loss and accumulation of toxic flaps that may then anneal forming bubbles in the DNA. Furthermore, it has been shown that abno rmal expression of Fen-1 may cause defective BER and thus, accumulation of mutations that can lead to genomic instab ility (Liu et al., 2004; Shibata and Nakamura, 2002). Lastly, another wa y in which Fen-1 may contribute to genomic stability is in its role in pr eventing tri-nucleotide repeat expa nsion and contraction. Studies in yeast have shown that mutations in Fen-1 lead to an expansion of trinucloeotide repeats through flap misalignment suggesting that Fen-1 is i nvolved in their prevention (Schweitzer and Livingston, 1998; White, 1999). Post Translational Mo difications of Fen-1 To date only two types of post translational m odifications have been identified for Fen-1: acetylation and phosphorylation. Both of these modifications have an effect on Fen-1s endonuclease and exonuclease activi ties. Acetylation of Fen-1 is accomplished through complex formation with the transcrip tional co-activator p300. Speci fically, Lys 354, Lys 375, Lys 377, and Lys 380 are acetylated in vitro and in vivo Acetylation of Fen-1 cau ses a reduction in the endonuclease and exonuclease act ivities of Fen-1 without affecting PCNA binding. Thus, acetylation may provide a means of regulating Fen-1 activity and as a result of Fen-1 down regulation, affecting DNA metabolic events (Hasan et al., 20 01; Henneke et al., 2003). 35

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Phosphorylation of Fen-1 also has an effect on its endonuclease and exonuclease activities. The phosphorylation of Fen-1 is mediated by cyclin dependent kinase (Cdk) 1-cyclin A complex. Phosphorylation occurs at Ser 187 wh ich is located in the internal nuclease domain. In contrast to acetylation, phosphorylation abolishes PCNA bindi ng, and thus PCNA-mediated stimulation of Fen-1 endonuclease and exonucleas e activities. However, phopshorylation does not affect the DNA binding activity of Fen-1. The phosphorylati on of Fen-1 by Cdk1-cyclin A suggests a critical role for phosphorylati on as a cell cycle regulatory mechanism for Fen-1 activity (Henneke et al., 2003). Purpose/Significance s previously stated, APC was able to interact with two BER proteins poland Fen-1 and interfere with pol-directed strand-displacement synthesis (Jaiswal et al., 2006; Narayan et al., 2005). Some cancer therapeutic drugs are DNA-alkylat ing agents such as 1,3-bis(2-chloroethyl)1-nitrosourea (BCNU) and temozolomide (TMZ) whic h create lesions that can be repaired by the BER pathway. Through a more complete understanding of APCs blockage of BER it may be possible to improve the efficacy of these chemothe rapeutic drugs. Another significance of these studies is in the basic science aspect of BER. As I discussed above, there are two types of BER pathways: SPand LP-BER. How these two pathways are selected by cells is not very clear. There is some evidence suggesting that the t ype of DNA damage is responsible for this distinction. Our studies suggest th at APC may play an additional role in distinguishing the SPversus LP-BER pathway along with the type of DNA damage present in the cell. By determining where the interaction of APC with poland Fen-1 is occurring, it wi ll be possible to elucidate the mechanism that APC is using to block BER. This knowledge will shed more insights into 36

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APCs involvement in BER and how to exploit this involvement to increas e the efficacy of DNA alkylating agents used as chemotherapeutic drugs. This study will focus on determining the interaction sites of APC with poland Fen-1 with particular emphasis on the APC and Fen-1 interaction and the mechanism by which APC is blocking BER through its interaction with Fen1. 37

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38 Figure 1-1. Roles of APC. Examples of the many cellular functions that APC has been implicated in are depicted in the above diagram. APC F-actin Microtubules Adhesion -catenin/TCF Apoptosis Migration Chromosome Se g re g ation c myc cdk4 Rb Proliferation Differentiation

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DRI-domain Figure 1-2. Structure of th e APC protein. Structure of the APC protein showing its -catenin binding and downregulation sites, its microtubule binding site, the mutation cluster region, and the DRI-domain. 39

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40 DNA base excision repair (BER) PathwaysReduced or oxidized AP site TGATC ACTAG T TGATC ACTAG T OH P APE1 TGATC ACTAG T OH Ppol(dRPaseactivity) TGATC ACTAG pol+ DNA ligase I or DNA ligase III/XRCC1A T Short-patch BER TGATC ACTAG T TGATC ACTAG T OH APE1 TGATC ACTAG T pol, polor poland PCNA/RF-C TGATC ACTAG FEN1 (DNaseI) and PCNA DNA ligase IA T Long-patch BER R P RTC AG TC AG TC AG TC AG P RA AT A C TGATC ACTAG T U UridineDNA Glycosylase Figure 1-3. Base excision repair. BER repair has two main pa thways- Short-patch BER and Long-patch BER.

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Figure 1-4. Structure of Fen-1. Diagram depicting important amino acids of Fen-1. Residues in dark blue show the amino acids involved in the active center of Fen-1. Residues shown in red are part of the seven loops important for substrate binding while residues shown in blue are important for post translational modifications. Amino acid residues shown in yellow are involved in th e PCNA interactions while those in green are involved in nuclear localization of Fen-1. Taken with permission from Shen et al., 2005. 41

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CHAPTER 2 MATERIALS AND METHODS Yeast Cell Maintenance The yeast strain PJ694-A was used for all yeast two-hybrid experiments. The yeast strain PJ69-4A ( MAT a trp1901 leu2 3,112ura352 his3 200 gal4 gal80 GAL2 ADE2 LYS2::GAL1 HIS3 met2 :: GAL7 lacZ ) was grown on synthetic dropout medium lacking lysine [(0.17% Difco yeast nitrogen base without amino acids, ammonium sulfate (5.0 g/L), complete supplemental amino acid mixture minus appropriate amino aci d containing 2% glucose)]. For long term storage the yeast cells were maintained in a glyc erol stock composed of yeast peptone dextrose media (YPD) and 40% glycerol and stored at -80oC. Yeast Two-Hybrid Assays of the Interaction of APC with Poland Fen-1 The yeast two-hybrid system was used to dete rmine the functional inte raction of APC with poland Fen-1 in vivo The APC DNA fragments containi ng the wild-type (amino acids 1190 1328) or the DRI-domain mutant (amino acids 1200 1324, in which amino acids Q1256, I1259, and Y1262 were replaced with alanine) for proteinprotein interaction stud ies were fused to the yeast Gal4 DNA binding domain (BD) in pl asmid pGBDU-C3. Single mutant APC cDNA fragments of the DRI-domain of APC(Q1256-A), APC(I1259-A), or APC(Y1262-A) were used to further characterize the poland Fen-1-interactions and also fused to the yeast Gal4-BD in plasmid pGBDU-C3. The interacting proteins such as full length poland Fen-1 were fused to the yeast Gal4 activation domain (AD) in plas mid pGADC3. Adapters were included as needed for the in-frame insertion of APC, pol, and Fen-1 DNA sequences relative to the Gal4-BD or Gal4-AD plasmids. The yeast strain S.cerevisiae PJ69-4A was co-transformed with pGBDU-C3 42

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and pGAD-C3 derived plasmids and spread on pl ates containing yeast synthetic dropout (SD) UL medium lacking only vector markers Ura for pGBDU-C3 derived plasmids and Leu for pGAD-C3 derived plasmids. To test for potential protein protein interactions, transformed cells were screened for growth on yeast SD ULH medium, which lacked Ura, Leu, and His, but contained 5 mM His3 inhibitor, 3-amino-1,2,4-triazole to prevent His3-reporter gene autoactivation. Maintenance of Mammalian Cells Human colon cancer cell lines HCT-116-APC( WT) (wild-type APC expression), HCT116 APC(KD) (knockdown APC expression by pSiRNA-AP C) were grown in McCoys 5a medium, and LoVo cells (truncated APC expression l acking DRI-domain) were grown in Hams F12 medium at 37 C under a humidified atmosphere of 5% CO2. In each case, the medium was supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 g/mL streptomycin. Generation of PolDeletion Constructs Plasmids were constructed to encode various segments of pol(amino acids 60 120, 80 170, 140200, and 160250) to identify th e amino acids of polthat interact with the DNA repair inhibitory (DRI) domain of APC. Thes e deletion fragments were subcloned into the pGAD-C3 vector between the Pst I and Bam HI restriction sites. The following primers were used to generate various poldeletion constructs: pol(60 120) (sense primer 5 CGCGGATCCAAGAAATTGCCTGGAGTA-3 and antisense primer 5 CCAATGCATTGGTTCTGCAGTTTAATTCCTTCATCTAC-3 ), pol(80 170) (sense primer 5 -CGCGGATCCGGAAAATTACGTAAACTG-3 and antisense primer 5 CCAATGCATTGGTTCTGCA GATCCACTTTTTTAACTT-3 ), pol(140200) (sense primer 43

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5 -CGCGGATCCCTGAAATATTTTGGGGAC-3 and antisense primer 5 CCAATGCATTGGTTCTGCAGG AAGCTGGGATGGGTCAG-3 ), and pol(160 250) (sense primer 5 -CGCGGATCCGATATTGTTCTAAATGAA-3 and antisense primer 5 CCAATGCATTGGTTCTGCAGAT ATTCTTTTTCATCATT-3 ). Site Directed Mutagenesis of PolTwo different sets of pol-mutants, Set-1 mutant (T79A/K81A/R83A) and Set-2 mutant (R89A/Q90A/D92A), were ge nerated by use of the Quick Change site-directed mutagenesis kit from Stratagene (La Jolla, CA). The following primer pairs were used for Set-1 and Set-2 mutants: Set-1, sense primer 5 GAAAAGATTGATGAGTTTTTAGCAGCCGGA GCGTTAGCTAAACTGGAAAAGATTC GGCAG-3 and antisense primer 5 CTGCCGAATCTTTTCCAGTTTAGCTAACGCTCCGGCTGCTAAAAACTCATCAATCTT TTC-3 ; Set-2, sense primer 5 GGAAAATTACGTAAACTGGAAAAGATTGCC GCGGATGCTACGAGTTCATCCATCA ATTTCCTG-3 and antisense primer 5 CAGGAAATTGATGGATGA ACTCGTAGCATCCGCGGCA ATCTTTTCCAGTTTACGTA ATTTTCC-3 Yeast Two-Hybrid Assay of PolDeletion Constructs, PolMutants, and APC The yeast two-hybrid assay was employed to identify critical amino acids of polto define the functional interaction with APC in vivo APC cDNA fragments containing the wildtype (residues 1190-1328) or the mutant DRI do main (residues 1200-1324, in which amino acids Ile1259 and Tyr1262 were replaced with alanine) were fused to the yeast Gal4 DNA-binding domain (BD) in plasmid pGBDU-C3. The interacting polprotein fragments such as full-length 44

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and various segments (residues 60-120, 80-170, 140-200, and 160-250) were fused to the yeast Gal4 activation domain (AD) in plasmid pGAD-C3. Set-1 (T79A/K81A/R83A) and Set-2 (R89A/Q90A/D92A) polmutants also were cloned into plasmid pGAD-C3. Yeast two-hybrid analysis was performed as described earlier. APC Peptides An APC peptide, 20-amino aci ds in length, (1250-KVSSINQET I QT Y CVEDTPI-1269) was synthesized at the Protein Ch emistry and Biomarkers core facility at the Interdisciplinary Center for Biotechnology Research (ICBR) of the University of Florida. They represent the DRIdomain of the wild-type APC (APCwt) or a muta ted form of the APC, APC(Q-A,I-A,Y-A), or APC(I-A,Y-A) in which amino acids Q1256, I1259, and Y1262 were replaced with alanine (A) (1250-KVSSIN A ETA QT A CVEDTPI-1269 or 1250-KVSSINQET A QT A CVEDTPI-1269). Synthesis and Labeling of the Fen-1 Substrate The Fen-1 substrates for 5-flap endonuclease activity were made by annealing an upstream 23-mer (5-CTAGATGCCTGCAGCTGATGC GC-3) and a downstream 45-mer or (5-FTTTTTGTACGGATCCACGTGTACGGTACC GAGGGCGGGTCGACA-3) or 51-mer (5-AACATTFTTTTTGTACG GATCCACGTGTACGGTACCG AGGGCGGGTCGACA-3) oligonucleotide to a 63-mer complement ary template (5' AGATGCCTGCAGCTGATGCG CGGTACGGATCCACGTGTACGGTACCGA GGGCGGGTCGACA-3'). The 45-mer downstream oligonucleotide has a flap of 5-nts while the 51-mer has a flap of 11-nts. Both flaps are cleaved by Fen-1. The Fen-1 substrate for 5-3exonuclease activit y was made similarly except that the 5-nt flap from the 45-mer oli gonucleotides was removed. After annealing with a 63-mer complementary template as described above, these oligonucleotides created a nick in the DNA suitable for Fen-1 exonuclease activity. The 45-, 51and 40-mer downstream oligonucleotides were radiolabeled at the 5-end with [-32P]ATP and T4 polynucleotide kinase 45

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(New England Bio Lab, Woburn, MA). The labeled probe was purified by using a nick column (GE Healthcare, Piscataway, NJ). All three oligo nucleotides were annealed at a molar ratio of 1:1:1. Fen-1 Activity in vitro The assays for 5-flap endonuc lease and 5-3 exonuclease activities for Fen-1 were performed in a final volume of 20 l. The reaction mixture cont ained 30 mM Hepes at pH 7.5, 30mM KCl, 8 mM MgCl2, 1 mM dithiothreitol (DTT), 200 g/mL bovine serum albumin (BSA), and indicated amounts of Fen-1 and APCwt or DRI-domain mutant APC [APC (I-A, Y-A)] peptides. After addition of the APC peptides, the reaction mixture was incubated at room temperature for 5 min. Then 2.5 nM 32P-labeled flapped-DNA or ni cked-DNA substrates were added to the mixture and furthe r incubated at 37C for 30 min. R eactions were terminated with stop solution containing 0.4% (w/v) SDS and 5 mM EDTA The DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation. The 5-nt or 11-nt DNA products from the 5-flap endonuclease activity and 1-nt product from the 5-3 ex onuclease activity were separated on a 15% acrylamide and 7 M urea ge l and quantitated by electronic autoradiography (InstantImager; Packard Instrument Co.,Meriden, CT ). Statistical analys is (n=3) was performed using a t-test on the Sigma Plot computer software. PCR of Fen-1 Protein Fragments for use in in vitro Transcription/Translation To identify the APC-binding site on Fen-1, di fferent Fen-1 cDNA constructs were made, Fen-1 protein fragments we re expressed using the in vitro transcription/translation system and then the interaction of APC and Fen-1 was analyzed by Far-Western Analysis. Primers for each protein fragment were designed to include a T7 promoter and a kozak sequence in the sense primer and a stop codon and poly A tail in the anti-sense primer. Five fragments of Fen-1 were 46

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designed (amino acids 1-115, 78-192, 154-268, 231-345, and 269-380). The following primers were used to generate the protein fragme nts: Fragment 1-115: (sense primer: 5 TAATTTTAATACGACTCACTATAGGGAACAGCCACCATGGGAATTCAAGGCCTGGCC 3 and anti-sense primer: 5TTTTTTTTTTTTTTTTTTTTTTTTTTTTTACT GAGCCTGCTGCAGC TGCTTCTCTGCCT C3), Fragment 78-192: (sense primer: 5TAATTTTAATACGACTCACTATGGGAAC AGCCACCATGGGCATCAAGCCCGTGTAT 3 and anti-sense primer: 5TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATCGCATT AGCACAGGGCTGCCGAACCT 3), Fragment 154-268: (sense primer: 5TAATTTTAATACGACTCACTATAGGGAACAGCCACCATGGATGCACCCAGTGAGG CA3 and anti-sense primer: 5TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAGGGTACTTGTTGGGGTCAAGTCGCA C3), Fragment 231-345: (sense primer: 5 TAATTTTAATACGACTCACTATAGGGAAC AGCCACCATGGGCAGTGACTACTGTGAG AGT3 and anti-sense primer: 5 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTT GAAGAAATCATCCA GGCGGCCCTG GGT3), and Fragment 269-380: (sense primer: 5 TAATTTTAATACGACTCACTATAGGG AACAGCCACCATGGTGCCAGAAAATTGGCTC CAC3 and anti-sense primer: 5 TTTTTTTTTTTTTTTTTTTTTTTATTTTCCCC TTTTAAACTTCCCTGCTGCCCCAGTCTTT GC3). 47

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The full length protein was synthesized using th e sense primer from 1-115 and the anti-sense primer from 269-380. The PCR of these fragme nts was carried out using Herculase, a combination of Pfu DNA polymerase and Taq po lymerase, according to the manufacturers recommendations (Stratagene, La Jolla, CA). Site-Directed Mutagenesis of Fen-1 Two sets of Fen-1 mutants (set 1: K354A/R355A/K356A and set2: K365A/K366A/K367A) were generate d using the Quick Change site directed mutagenesis kit (Stratagene, La Jolla, CA). To generate each mu tant two pairs of primers were used. One pair was used first to generate two amino acid mutati ons. Then the resultant mutant protein was used as template and other primer pair was used to generate the last amino acid mutation. The following primer pairs were used to generate the set 1 and set 2 mutants: Set-1 primer pair 1, sense primer: 5 GGCTCACTCTCTTCAGCTGCGCGCGCAGAGCCAGAACCCAAGGGATCC3 and antisense primer: 5 GGATCCCTTGGGTTCTGGCTCTGCGCGCGCAG CTGAAGAGAGTGAGCC3 and Set-1 primer pair 2, sense primer: 5 GGCTCACTCTCTTCAGCTGCGGCCGCAGAGCCAGAACCCAAGGGATCC3 and antisense primer: 5 GGATCCCTTGGGTTCTGGCTCTGCGGCCGCAGCTGAAGAGAGTGAGCC3; Set-2 primer pair 1 sense primer: 5 GAACCCAAGGGATCCACTGCGAAGGCCGCAAAGA CTGGGGCAGCA3 and anti-sense primer: 5TGCTGCCCCAGTCTTTGCGGCCTTCGCAGTGGATCCCTTGGGTTC3; Set-2 primer pair 2, sense primer: 5 48

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GAACCCAAGGGATCCACTGCGGCAGCCGCAAAGAC TGGGGCAGCA3 and anti-sense primer 5 TGCTGCCCCAGTCTTTGCGGCTGCCGCAGTGGATCCCTTGGGTTC3. Synthesis of Fen-1 Protein Fragments, Fen1 wt, and Fen-1 Mutant Proteins Using in vitro Transcription/Translation Full-length Fen-1, Fen-1 prot ein fragments, and Fen-1 set-1 and set2 mutant proteins were synthesized using the TNT PCR Quick Kit from Promega (Madison, WI). PCR product was added directly to the rabbit reticulocyte mix al ong with the methionine provided with the kit and 50 M MG132. The reaction was run as per instructions provided with th e TNT PCR Quick Kit from Promega. Synthesis of Fen-1 proteins a nd Fen-1 protein fragment s was determined by 15% SDS-PAGE analysis followed by western blottin g. Fen-1 proteins and pr otein fragments were detected using an anti-mouse Fen-1 antibody (Novus Biologicals, Littleton, CO) that was able to detect amino acids 1-380 of Fen-1. The signals were detected using the chemiluminescence technique (GE Healthcare, Piscatway, NJ). Far-Western Blot Analysis APCwt and APC(I-A,Y-A) peptid es were slot-blotted (0-15 g) onto a polyvinylidene difluoride membrane (GE Healthcare, Piscatway, NJ) in a binding buffer containing 20 mM TrisCl at pH 7.4, 100 mM phosphate buffer at pH 7.4, 60 mM KCl, and 0.25% (v/v) Nonidet P-40. After blotting, the membrane was blocked with 5% (w/v) bovine serum albumin and washed three times with Tris-buffere d saline with 0.025% (v/v) Tween20 prior to incubation with in vitro transcription/translation synthe sized Fen-1 protein fragments (described above), and wild type, K354A/K355A/K366A mutant or K365A/K366A/K367A muta nt human Fen-1 proteins. Binding was detected using mouse anti-Fen-1 an tibody (Novus Biologicals, Littleton, CO). The signals were detected using the enhanced chemiluminescence technique (GE Healthcare, Piscatway, NJ). 49

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Pull-Down of Fen-1 wild type and Fe nS-1 K365A/K366A/K367A from TNT Synthesis Reaction Mixture Fen-1 wild-type (Fen-1wt) and Fen-1 K365A/K 366A/K367A mutant (Fen-1 mutant) were pulled-down by incubating them with rabbit an ti-human Fen-1 antibody in immuno-precipitation (IP) buffer (30 mM Hepes at pH 7.5, 30 mM KCl, 8 mM MgCl2, 0.01% NP-40, 2% Glycerol) for 4 hours followed by Protein A sepharose beads (Promega, Madison, WI), that were blocked with 5% BSA (v/v), for an additional 2-3 h. Beads we re washed with washing buffer (30 mM Hepes at pH 7.5, 30 mM KCl, 5% glycerol) 3 times and then beads were resuspended in reaction buffer and used in Fen-1 activity assays. The beads were then used in the indicated amount for the Fen1 activity assays. Immunohistochemistry Human colon cancer cell lines LoVo, HCT116-APC(WT), and stable knockdown HCT116-APC(KD) were plated into 6-well plates c ontaining sterilized cover slips. Each well contained about 80-100,000 cells. Ce lls were allowed to grow for 48 h and were then treated with 500 M MMS for 24 h and fixed using 4% Paraformaldehyde in phosphate-buffered saline (PBS). For time course experiments, cells were treated with 500 M methylmethane sulfonate (MMS) for 5, 15, or 24 h. At the appropriate time point cells were fixed using 4% Paraformaldehyde in PBS. Once fixed, cells were quenched using 20 mM ammonium chloride in PBS. Fixed cells were then incubated in mouse anti Fen-1 antibody (Abcam, Cambridge, MA) and rabbit anti APC antibody (Santa Cruz, Santa Cr uz, CA) for 2hrs. The fixed cells were then washed four times for 5 min. in PBS and were su bsequently incubated in anti mouse Alexa Flour 488 and anti-rabbit Alexa Flour 595 antibodies (Invitrogen, Carlsbad, CA) for 1 h. Cells were washed as previously described and were mount ed onto slides using Vecta Shield (Vector 50

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51 Laboratories, Burlingame, CA). Slides were viewed under a Zeiss Upright Microscope at wavelength 500 nm for Fen-1 staini ng and 640 nm for APC staining. Western Blot Analysis of Fen1 and APC Protein Levels in the Cytoplasm and the Nucleus in Cells Treated with DNA-Alkylating Agents HCT-116-APC(WT) and HCT-116-APC(KD) cells were treated with 500 M methylmethane sulfonate (MMS) for 5, 10, 15 or 25 h. Nuclear and cytosolic fractions from MMS-treated HCT-116-APC(WT) and HCT-116-APC(KD) cells were prepared as described in (Shapiro et al., 1988). In order to determine Fen1 levels in the nucleus and the cytoplasm, 15 g of the nuclear and cytosolic fractions were analyzed on a 10% SDS-PAGE followed by Western blotting. A mouse anti-Fen-1 antibody (Novus Biologi cals, Littleton, CO) was used to detect the presence of Fen-1 in the nuclear and cytosolic fractions. The signals were then detected using the chemiluminescence technique (GE Healthcare, Pi scataway, NJ). In order to determine APC levels in the nucleus and the cytoplasm, 50 g of the nuclear fractions and 80 g of the cytosolic fractions were analyzed on a 4% SDS-PAGE followed by Western blotting. The presence of APC was detected using a mouse anti-APC anti body (Millipore, Temecula, CA). The signals were then detected using the chemiluminescence technique (GE Healthcare, Piscataway, NJ).

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CHAPTER 3 APC INTERACTS WITH DNA POLYMERASE BETA Amino acids Thr79/Lys81/Arg83 of polare Critical for its Interaction with APC Previous studies have shown that APC and polinteract in vivo and that this interaction blocks pol-directed strand-displacement synthesis (Nar ayan et al., 2005). In order to map the critical amino acids of polnecessary for its interaction with APC, deletion constructs of polwere generated through PCR am plification and cloned into the pGAD-C3 yeast two-hybrid (Y2H) vector. A total of four deletion construc ts were made (Figure 3-1A). These constructs were then used in a Y2H assay along with APC w ild-type protein (APCwt). As seen in Figure 31B, the deletion constructs containing amino aci ds 60-120 and 80-170 were able to interact with APCwt while with the APC DRI-domain muta nt [APC(I-A, Y-A)] construct there was no interaction. Known interactions of wild-type APC and polPCNA and polwere used as positive controls, while the pol-activating domain was used as a negative control for background colonies. Screening fo r interacting proteins was pe rformed as described in the Materials and Methods. In order to further define the important ami no acids from the interacting region of amino acids 80-120, we examined solvent surface accessibility of the residues implicated in the interaction with APC. Since a complete structur e of APC does not exist, it was not possible to identify possible interactions through docking modes. Intere stingly, amino acids 80-120 are located in a region that connect s the 8 kDa lyase domain and th e 31 kDa polymerase domain of polFrom the structure of the ternary substrate complex two regions set-1 (amino acids Thr79, Lys81, and Arg83) and set-2 (amino acids Arg89, Gln90, and Asp92) were identified that exhibited high solvent a ccessibility (Fig. 3-2). 52

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Site-directed mutagenesis was used to change each set of amino acids to alanine and generate two polmutants. One mutant contains the ami no acids of set-1 muta ted to alanine and the other contains the amino acids of set-2 mutated to alanine. The two polmutants were then cloned into the Y2H vector pGAD-C3 and a Y2H analysis with APCwt was used to determine if either set of mutants was able to disrupt the interaction between poland APC. As seen in Figure 3-3, the polset-1 mutant was able to disr upt the interaction while the polset-2 mutant was unable to interrupt the inte raction. This indicates that am ino acids Thr79, Lys81, and Arg83 are critical for the interaction of polwith APC. Amino Acids I1259A and Y1262A, but not Q1 256A are Required for APCs Interaction with DNA Polymerase (Pol) It was originally thought that APC contained a PCNA intera cting protein (PIP)-like box composed of Q1256, I1259, and Y1262 and all three amino acids were necessary for the interaction of APC with polIn order to test this possibility site-directed mutagenesis of the APCwt fragment used previously (Narayan et al., 2005) was carried out. Single mutation of each amino acid to alanine was carried out and each muta nts ability to disrupt the interaction of APC with the proteins was analyzed via Y2H assa y (Fig. 3-4). After each single mutant was constructed, the peptide was cloned into the Y2H vector pGBDU-C3. Full length polwas already in the Y2H pGAD-C3 vector. Screening for interacting proteins was conducted in the same manner as described in the Materials and Methods. The effects that the single APC mutants Q1256A, I1259A, and Y1262A had on the abil ity of APC to interact with polcan be seen in Figure 3-3B. The single mutant Q1256A cannot impede the interaction between poland APC. However, the single mutants I1259A and Y1262A can disrupt the ability of APC to interact with polindicating that Y1259A and Y1262A are equally importan t in the ability of APC to interact 53

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with polThese findings indicate that the PIP-like box found in APC is actually not a PIP-like box. In order to be a PIP-like box al l three amino acids involved must have been involved in the interaction. Clearly, this is not the case for APC. The PIP-lik e box was therefore renamed the D NA R epair I nhibitory (DRI)-domain after much consid eration. Known intera ctions of wildtype APC and pol, PCNA and pol, and Daxx and p53 were used as positive controls, while the APC binding domain was used as a negative control for background colonies. 54

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B. Yeast two-hybrid analysis A. Deletion constructs of Pol0 335 aa 60 120 aa 80 170 aa 140 200 aa 160 250 aaP o l W tP C N A + P o l P o l W t + A P CP o lW t + A P C ( I A Y A )P o l ( 6 0 1 2 0 ) + A P CP o l ( 6 0 1 2 0 ) + A P C ( I A Y A )P o l ( 8 017 0 ) + A P CP o l ( 1 6 0 2 5 0 ) + A P C ( I A Y A )P o l ( 1 6 0 2 5 0 ) + A P CPo l ( 8 0 1 7 0 ) + A P C ( I A Y A )P o l ( 1 4 0 2 0 0 ) + AP CP o l ( 1 4 0 2 0 0 ) + A P C ( I A Y A ) KAATCKVSSINQET QT CVEDTPI 1324 aa 1190 1245 1269APC Pol12123 4 5 6 7 8 9 10 11 1259 1262 Figure 3-1. Interaction of APC with pol-deletion constructs. A) De letion constructs of polB) Y2H assay of pol-deletion constructs and APC; inte ractions used are depicted in the diagram. Taken with permission from Balusu et al., 2007. 55

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Figure 3-2. Ribbon representation of polhighlighting the position of key mutants. A) Set 1 (red) and Set 2 (purple) resi dues are displayed of a ternar y substrate complex of pol. Lyase domain is shown in gold and the pol ymerase domain is shown in blue (PDB accession code 2MFS). The DNA backbone is s hown in orange while a light blue sphere (Mg2+) identifies the polymerase activ e site and a red sphere (NZ of lyase 72) identifies the lyase active site. B) Set 1 (residues 79-84) side chains. C) Set 2 (residues 87-92) side chains. Taken with permission from Balusu et al., 2007. 56

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APC Pol-Wt APCAPC + PolMut-2APC + PolMut-1PCNA + Pol-Wt B. Yeast two-hybrid analysis A. Structure of peptidesAEKIDEFLATGKLRKLEKIRQDDTSSS 335 aa 0 AEKIDEFLA G L KLEKIRQDDTSSS AEKIDEFLATGKLRKLEKI D TSSS 335 aa 0 335 aa 0 PolWt PolMut-1 PolMut-2 No experiment1 2 3 4 5 6 8183 79 8183 79 9092 89 9092 89 Figure 3-3. Interaction of APC with polmutants. A) Structure of the peptides used in the Y2H assay. B) Y2H assay of the two polmutants and APC; interactions used are depicted in the diagram. Adapted with permission from Balusu et al., 2007. 57

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APC(Q-A, I-A,Y-A) polAPCwt polAPC(Q-A) polpolwt APC(Y-A) polAPC(I-A) polAPCwt(1190-1328) 1250-KVSSIN ET QT CVEDTPI-1269 APC(Q-A,I-A,Y-A) KVSSIN ET QT CVEDTPI APC(Q-1256A) KVSSIN ET QT CVEDTPI APC(I-1259A) KVSSIN ET QT CVEDTPI APC(Y1262A) KVSSIN ET QT CVEDTPIA. Structure of the APC peptides B. APC/polinteraction Figure 3-4. Interaction of APC mutants with pol. A) Structure of the peptides used in this assay. B) Y2H assay of APC mutant proteins with pol, interactions used shown in the diagram. Adapted with permission from Jaiswal et al., 2006. 58

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CHAPTER 4 APC INTERACTS WITH FLAP ENDONUCLEASE 1 (FEN-1) APC and Fen-1 interact in vivo APC and Fen-1 were shown to physically interact by immuno-precipitation and Farwestern analysis (Jaiswal et al., 2006). However, this needed to be confirmed in vivo In order to accomplish this, a Y2H assay was used. Full length Fen-1 that was already cloned into the Y2H vector pGAD-C3, APCwt fragment which contains the DRI-domain (amino acids 1190-1328), and APC mutant (mut) fragment which contai ns a mutated DRI-domain (amino acids 1200-1324 with 1259I-A, and 1262Y-A mutatio ns) that was already cloned into the Y2H vector pGBDUC3 were used to run the Y2H assay. As Figure 4-1 clearly shows, APCwt is able to interact with Fen-1 in vivo The known interaction of PCNA and polwas used as a positive control, while the APC binding domain was used as a negative control to check for background colonies. Lys-, Leu-, Ura-, His-selection media was used to screen for interacting col onies. In order to grow, the yeast cells needed to make histidine and the only available method to do so is for the two proteins to interact. The Interaction Site of APC is the Same for Fen-1 and PolOnce the interaction s ite of APC and polwas determined, it was thought that the same amino acids that are necessary for APCs interaction with polmay be important for APCs interaction with Fen-1 since APC interacts with Fen-1 at the DRI-domain. In order to determine this, a Y2H analysis was carried out with Fen1 and the single mutant s of APC that were previously used in the yeast two-hybrid analysis with pol. It was found that the single mutants I1259A and Y1262A of APC were able to di srupt the interaction of APC and Fen-1. 59

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However, the Q1256A single mutant of APC did not have an effect on th e interaction of APC and Fen-1 (Fig. 4-2). These results are identical to that of the interaction site of APC and polindicating that APC interacts with thes e BER proteins at the same site. APC Blocks the Endonuclease and Exonuclease Activi ties of Fen-1 Since APC was found to interact with Fen-1 in vivo and in vitro, the effect of this interaction on the catalytic activities of Fen1 was measured. Fen-1 contains two types of cleavage activity that are rele vant for completion of BER: 5 endonuclease activity and 53exonuclease activity. The 5 endonuclease activity is important in strand-displacement during LP-BER where it removes the flap, while the 5-3 exonuclease activity is important for Hitand-Run synthesis of LP-BER. In hit-and-run s ynthesis, Fen-1 excises a nucleotide at the 3side of a nick to create a gap to be filled by pol(Klungland and Lindahl, 1997; Prasad et al., 2001; Qiu et al., 2001; Shibata and Nakamura, 2002). In order to test the effect of APC on Fen-1 cleavage activity, three in vitro assays were run: two which used substrates specific for the 5 endonuclease activity of Fen-1 and one that used a substrate specific for the 5-3 exonuclease activity of Fen-1. To test APCs effect on Fen1 5 endonuclease activity, an in vitro 5 flap endonuclease assay in the presence or abse nce of APC peptide (20 amino aci ds long with DRI-domain in the middle) using a 32P-labeled flapped substrate was conducted. As can be seen in Figures 4-3 and 4-4, cleavage of the substrate by Fen-1 results in a 5 or 11 nt product. When the APCwt peptide is added cleavage of the flap by Fen-1 is blocked in a dose-de pendent manner (compare lane 2 with lanes 3-7 in both Fig. 4-3A and Fig. 4-4A, respectively). 60

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However, when the APC(I-A,Y-A) mutant peptide is used cleavage of the flap by Fen-1 appears to be unaffected (compare lane 2 with lanes 8-12 in both Fig. 4-3A and Fig. 4-4A, respectively). A quantitative analysis of the data is shown in Figure 4-3B and Figure 4-4B. In order to determine the effect of APC on Fen-1 5-3exonuclease activity, a nicked DNA substrate was used. The nicked DNA substrate was formed in the following manner: a 23-mer upstream and 40-mer downstream oligonucleotides were annealed with a 63-mer complimentary template oligonucleotide as de scribed in the materials and methods section. The 40-mer oligonucleotide was labeled with 32P[ATP] before annealing at the 5end. The 5-3 exonuclease activity of Fen-1 will cleave the nick re sulting in a 1-nt product. The results show a 1-nt cleavage product when Fen-1 is added to the substrate. However, in the presence of APCwt peptide cleavage of the nicked substrate by Fen1 is blocked in a dose-dependent manner (Fig. 45A compare lane 2 with lanes 3-6). The 5-3 exonuclease activity of Fen-1 appears to be unaffected in the presence of the APC(I-A,Y-A) mu tant peptide (Fig. 4-5A compare lane 2 with lanes 7-10). A quantitative analysis of the data is shown in Figure 4-5B. Amino Acids K365/K366/K367 are Important fo r the Interaction of Fen-1 with APC In order to determine which amino acids of Fe n-1 were important for its interaction with APC, five different prot ein fragments of Fen-1 were synthesized using the transcription coupled translation (TNT) Quick PCR kit from Promega (M adison, WI). The five protein fragments are depicted in Figure 4-6A. Synthesis of the prot ein fragments was determined via Western blot analysis. As seen in Figure 4-6B, all protein fr agments as well as Fen-1wt were detected. Equal amounts of proteins were estimated by analyzing the densities of the bands from the Western blot. A non-specific band was seen with all e xpressed Fen-1 fragments, which was originating due to the cross-reactivity of antiFen-1-antibody with TNT extract. 61

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Far-western analysis was then performed us ing the Fen-1 protein fragments and APCwt and APC(I-A,Y-A) mutant peptides to determine where the interaction of Fen-1 with APC was occurring. The only Fen-1 protein fr agment able to interact with the APCwt peptide was the Fen1 protein fragment which cont ained amino acids 269-380 indicati ng that this region of Fen-1 might be responsible for the in teraction with APC (Fig. 4-7). The overlapping Fen-1 protein fragment which contained amino acids 231-345 was not able to inte ract with the APCwt peptide indicating that the region of Fen-1 that inte racts with APC is betw een amino acids 346-380. The interacting site was further narrowed dow n by analyzing the role of amino acids 346380 in the structure and function of Fen-1, and by re viewing the literature fo r other proteins that interact with Fen-1 in this region. The most striking thing ab out amino acids 346-380 was that most of the amino acids are a part of the nuclear localization signal of Fen-1 (Shen et al., 2005). Furthermore, several proteins have been found to interact with Fen-1 at the C-terminus. Interestingly, three of the proteins were found to interact with Fen-1 between amino acids 353380 which overlaps with the region of Fen-1 dete rmined to interact with APC from the Farwestern analysis (Guo et al., 2008; Sharma et al., 2005). Based on the above information two sets of Fen-1 amino acids (K354/R355/K356 and K365/K366/K367) were chosen to mutate to see if they were important for the interaction of Fen-1 with APC. Each set was chosen based on its importance in nuclear localization and on the f act that the amino acids chosen were in the range identified for the interaction of Fen-1 with other proteins such as Werner protein (WRN), Blooms syndrome protein (BLM), and endonucleas e G (EndoG). Each amino acid was mutated to alanine and the two Fen-1 mutants were s ynthesized using the TNT PCR Quick Kit from Promega (Madison, WI). Synthesis of the Fen-1 mutants was determined using Western blot analysis. As seen in Figure 4-8, Fen-1wt and mutant proteins were present after synthesis. Equal 62

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amounts of proteins were estimated as describe d for the Fen-1 protein fragments and the Fen1(K354A/R355A/K356A) and Fen1(K365A/K366A/K367A) mutants were used in a Farwestern analysis. Both Fen-1(K354A/R 355A/K356A) and Fen-1( K365A/K366A/K367A) mutants were able to decrease the bind ing of APC and Fen-1. However, the Fen1(K365A/K366A/K367A) mutant was able to decrease binding more than the Fen1(K354A/R355A/K356A) mutant in dicating that the Fen-1(K365A/K366A/K367A) mutant is more important for binding (Fig. 4-9A). Percent bi nding of the Far-western analysis can be seen in Figure 4-9B. Fen-1(K365A/K366A/K367A) Mutant Retains Catalytic Activity Due to the fact that the Fen-1(K365A/K366A/K367A) mutant was able to reduce the binding of APC with Fen-1 and that previous expe riments had shown that APC was able to block Fen-1 catalytic activty, it was decided to investigate whether the Fen-1(K365A/K366A/K367A) mutant contained catalytic activity. In order to test the 5endonucleas e activity of the Fen-1 (K365A/K366A/K367A) mutant, a 32P-labeled flapped substrate was used as previously described in the Results sections. In order to use the TNT synthesized Fen-1wt and Fen1(K365A/K366A/K367A) mutant for the assays, the proteins were pulled-down from the TNT reaction mixture using an anti-Fen-1 an tibody. The beads with bound Fen-1wt or Fen-1 (K365A/K366A/K367A) mutant were then used in the endonuclease reaction. Endonuclease activity of the Fen-1wt a nd Fen-1(K365A/K366A/K367A) muta nt was determined by the presence of a 5-nt product. Interestingly, the Fe n-1(K365A/K366A/K367A) mu tant was able to cleave the 5 endonuclease substrat e as efficiently as the wild-t ype protein (Fig. 4-10C compare lanes 7-9 with lanes 10-12) indicating that mutations in Fen-1 at amino acids K365/K366/K367 are not important for the e ndonuclease activity of Fen-1. 63

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Additionally, the exonuclease activity of the Fen-1(K 365A/K366A/K367A) mutant was also tested. In order to determine the effect of APC on Fen-1 5-3exonuclease activity, a nicked DNA substrate was used. Labeling of the nicked substrate with -32P[ATP] was done as previously described in the Material s and Methods section. Fen-1wt and Fen1(K365A/K366A/K367A) mutant proteins were pulled-down from the TNT reaction mixture as described for the endonuclease activity. The beads with bound Fen-1wt or Fen1(K365A/K366A/K367A) mutant were then used in the exo nuclease reaction. Exonuclease activity of the Fen-1wt a nd Fen-1(K365A/K366A/K367A) muta nt was determined by the appearance of a 1-nt product. Surprisingly, the Fen-1(K365A/K366A/K36 7A) mutant had more exonuclease activity that the wild -type protein (Fig. 4-10D compar e lanes 4 and 5 with lanes 6 and 7). This could be due to the fact that the mutations may have caused a conformational change within the protein that may have caused the active site to be more accessible. Combined, the results from the endonuclease and exonuclease a ssays indicate that the mutations in Fen-1 at amino acids K365/K366/K367 are not important for the catalytic activity of Fen-1. Therefore, the blockage of Fen-1 activity by APC after interaction with ami no acids K365/K366/K367 could be due to the conformational change of Fe n-1 structure affecting its activity. Alternatively, it could be due to steric hindrance caused by th e APC protein since it is such a large protein. APC Prevents the Entry of Fen-1 into th e Nucleus after Exposure to DNA-Alkylating Agents The fact that the Fen-1(K 365A/K366A/K367A) mutant still c ontained catalytic activity prompted the idea that APC could be blocking Fen-1s participation in LP-BER through more than one mechanism. Since the Fen-1(K365A/K 366A/K367A) mutant does not affect catalytic activity, the purpose of an interaction with APC at this site remained unclear. In order to get a better idea of the functional in teraction of APC with Fen-1 at amino acids K365/K366/K367 an 64

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immunohistochemical analysis was performed to de termine the localization of these two proteins in cells after exposure to the DNA-alkylating ag ent, methylmethane sulfonate (MMS). In 2007, Kundu and colleagues, showed that the treatment of cells with M of MMS caused the upregulation of APC and that th is upregulation of APC caused a blockage of DNA repair. Based on these data, HCT-116-APC(WT) cells were treated with 500 M MMS to ensure that APC upregulation would occur and that it would interact with Fen-1. HCT-116-APC(WT) cells contain wild-type APC. Particular attention was paid to the local ization of APC and Fen-1 with regards to the nucleus as DNA repair occurs in the nucleus and the identified APC interaction site on Fen-1 occurs in the nuclear localizat ion signal. After 24 h of treatment with 500 M MMS, an interesting phenomenon can be seen in the HCT-116-APC(WT) cells. Fen-1 nuclear localization is severely decreased while the cytoplasmic level of Fen-1 has increased as compared to control HCT-116-APC(WT) cells. Mo reover, when the staining of Fen-1 (green) and APC (red) is merged a considerable amount of co-localization, as demarcated by a yellow color, is seen between Fen-1 and APC in the cy toplasm and more specifically around the nuclear membrane as compared to control HCT-116-APC( WT) cells (Fig 4-11). Interestingly, this phenomenon was not seen in HCT-116-APC(KD) cells subjected to the same treatment as the HCT-116-APC(WT) cells. The HCT-116-APC(KD) cell line has been stably transfected with siRNA against APC. Thus, APC levels are greatly reduced in this cell line. As seen in Figure 412, after 24 h of treatment the HC T-116-APC(KD) cells still contain the majority of Fen-1 in the nucleus indicating that APCs inte raction with Fen-1 may be playi ng a role in the loss of nuclear Fen-1 in response to treatment with MMS. In order to gain a more complete understanding of what was occurring during the 24 h treatment with MMS, a time course experiment was performed. The cells were treated with 500 65

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M MMS for a time period of 5, 15, or 24 h. In addition to the aforementioned cell lines HCT116-APC(WT) and HCT-116-APC(KD), the LoVo cell li ne was also used. LoVo cells were used because they contain truncated APC (120 kDa) wh ich lacks the DRI-domain and will not be able to interact with Fen-1. Thus, these cells serve as an additional method to ensure that the loss of nuclear Fen-1 after treatment with MMS for 24 h is dependent upon the APC and Fen-1 interaction. As expected, both LoVo cells and HCT-116-APC(KD) cells re tained nuclear Fen-1 throughout the length of the time course expe riment (Fig. 4-12 a nd 4-13). Moreover, colocalization (yellow) was not s een in the merged photographs of APC (red) and Fen-1 (green). On the other hand, a significant difference was observed between HCT-116-APC(WT) cells treated with MMS versus control cells as the time course experiment progressed. After 5 h of treatment, Fen-1 (green) is still localized in the nucleus and some Fen-1 is present in the cytoplasm while, APC (red) is seen in both the cy toplasm and the nucleus (Fig. 4-13). When the localization of APC (red) and Fen1 (green) are merged, more co-lo calization (yellow) is seen in both the cytoplasm and the nucleus of the HCT-1 16-APC(WT) cells as comp ared to control cells (Fig. 4-14), indicating that in re sponse to DNA damage there is an increased interaction between Fen-1 and APC. After 15 h of MMS treatment, about 50 percent of cells have developed a loss of nuclear Fen-1 (green) and an increase of cytoplasmic Fen-1 as compared to cells treated for only 5 h and HCT-116-APC(WT) control cells. Furtherm ore, APC (red) is localized more in the cytoplasm than in the nucleus. When APC (red) and Fen-1 (green) localization is merged a considerable amount of cytoplasmic co-localiz ation (yellow) is s een. Interestingly, colocalization can also be seen in the nucleus where the remaining Fen-1 is present (Fig. 4-12). After 24 h of treatment, most of the cells have developed a loss of nuclear Fen-1 (green) and an increase in cytoplasmic Fen-1 as compared to c ontrol cells and cells trea ted with MMS for only 5 66

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h. Furthermore, most of the APC (red) appears to be localized in the cytoplasm as compared to the nucleus although some nuclear APC also re mains in HCT-116-APC(WT) cells (Fig. 4-12). Lastly, when the localization of Fen-1 (green) a nd APC (red) is merged a significant amount of co-localization (yellow) is seen in the cytoplasm especia lly around the nuclear membrane. Moreover, some co-localization ca n still be seen between the re maining nuclear Fen-1 and APC, which is much lower than the interaction betwee n Fen-1 and APC which are co-localized in the cytoplasm after 24 h of MMS treatment (Fig. 4-12). Thus, it appears that APC is required for the loss of nuclear Fen-1 over time since neither Lo Vo cells nor HCT-116-AP C(KD) cells exhibited this phenomenon (Fig. 4-12 to 4-13). This discov ery provides a role for the interaction of APC with Fen-1 at its nuclear local ization signal by suggesting that APC prevents Fen-1 from entering the nucleus after exposure to DNA-damaging agents. In order to confirm the imm unofluorescence results, nuclear and cytosolic levels in HCT116-APC(WT) and HCT-116-APC(KD) cells treated with 500 M MMS for 5, 10, 15, and 25 h were determined by Western blot analysis. The cytosolic fraction showed no significant change in the levels of Fen-1 in both HCT-116-APC( WT) cells and HCT-116-APC(KD) cells treated with 500 M MMS (Fig. 4-14) while the nuclear fracti on showed a decrease in the levels of Fen-1 in MMS treated cells over time in HC T-116-APC(WT) when compared to control HCT116-APC(WT) cells (Fig. 4-14). However, no signif icant change was seen in the levels of nuclear Fen-1 in MMS treated HCT-116-APC(KD) ce lls (Fig. 4-15). These results are consistent with the findings of the immunofluorescence stud y, which showed similar results for nuclear Fen-1. Analysis of the APC levels in the cytoso lic and nuclear fractions showed an increase in the level of APC in MMS-treated HCT-116-APC(WT ) cells over time. As expected, APC levels were undetectable in MMS treated HCT-116-APC( KD) cells in both the nuclear and cytosolic 67

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fractions. Furthermore, the increase in cytoplasmi c APC correlates with th e decrease in nuclear Fen-1 suggesting that the cytoplasmic APC is preventing Fen-1 translocation into the nucleus. In HCT-116-APC(WT) cells treated with MMS for 15 h cytoplasmic APC reaches its highest levels while nuclear Fen-1 reaches its lowest levels At the 25 h time point, the cytoplasmic APC begins to decrease while nuclear Fen-1 shows a modest increase which further supports the hypothesis that APC may be bloc king nuclear translocation of Fen-1 in response to DNAalkylating agents. 68

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Figure 4-1. Interaction of Fen-1 and APC. Y2H assay showing the in vivo interaction of APCwt and Fen-1wt. A) Structure of the peptides used in the Y2H assay. B) Y2H assay of APC and Fen-1. Interactions used are shown in the diagram. 69

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70 APC(Q-A, I-A,Y-A) Fen-1APCwt Fen-1APC(Q-A) Fen-1APC(Y-A) Fen-1 APC(I-A) Fen-1 p53 DAXX APCwt PCNA polAPCwt(1190-1328) 1250-KVSSIN ET QT CVEDTPI-1269 APC(Q-A,I-A,Y-A) KVSSIN ET QT CVEDTPI APC(Q-1256A) KVSSIN ET QT CVEDTPI APC(I-1259A) KVSSIN ET QT CVEDTPI APC(Y1262A) KVSSIN ET QT CVEDTPIA. Structure of the APC peptides B. APC/Fen-1 interaction Figure 4-2. Interaction of APC mu tants with Fen-1. A) Structure of the peptides used in this assay. B) Y2H assay of APC mu tant proteins with Fen-1, in teractions used shown in the diagram. Adapted with permission from Jaiswal et al., 2006.

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Figure 4-3. APC blocks the 5-flap endonucleas e activity of Fen-1. A) Autoradiogram of a representative experiment. Reaction mixtures in a 25 L final volume contained a fixed (0.1 nM) concentrations of Fen-1 c oupled with different concentrations (0.2, 0.5, 1.0, 1.6, and 2.0 M) of either APCwt or mutant APC(I-A,Y-A) peptides. The mixture was incubated for 5 min on ice, and then 2.5 nM of the 32P-labeled flappedDNA substrate was added. The cleavag e reaction was carried out at 37oC for 30 min. X-ray film autoradiography determined the 5-nt cleaved product, and the electronic autoradiography measured the radioactivity (InstantImager; Packard Instrument Co., Meriden, CT). B) Quantitative analysis of the 5-mer cleaved product. The fraction of cleavage was calculated as the percent radio activity present in th e cleaved product as follows: % cleavage = [5-nt/(45-mer + 5nt)] -100. Data are the mean SE of three different experiments. 71

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Figure 4-4. APC blocks the 5-f lap endonuclease activity of Fen-1 regardless of flap size. A) Autoradiogram of a repres entative experiment. Reaction mixtures in a 25 L final volume contained a fixed (0.1 nM) concentra tions of Fen-1 coupled with different concentrations (0.2, 0.5, 1.0, 1.6, and 2.0 M) of either APCwt or mutant APC(IA,Y-A) peptides. The mixture was incubated for 5 min on ice, and then 2.5 nM of the 32P-labeled flapped-DNA substrate was adde d. The cleavage reaction was carried out at 37oC for 30 min. X-ray film autoradiography determined the 5-nt cleaved product, and the electronic autoradiography measur ed the radioactivity (InstantImager; Packard Instrument Co., Meriden, CT). B) Quantitative analysis of the 11-mer cleaved product. The fraction of cleavage wa s calculated as the percent radioactivity present in the cleaved produc t as follows: % cleavage = [11-nt/(45-mer + 11-nt)] 100. Data are the mean SE of three different experiments. 72

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Figure 4-5. APC blocks the 5-3 exonuclease activity of Fen-1. Exonuc lease activity reaction mixture was assembled in a total of 25 L final volume and contained fixed (15 nM) concentrations of Fen-1 along with (3, 5.8, 7.3 or 8.8 M of either APCwt or APC(IA,Y-A) peptides. The mixture was incubate d for 5 min at 22C and then 2.5 nM 63mer oligonucleotide substrates containing the 32P-labeled 40-mer downstream oligonucleotides were added. The exonuclease reaction was carried out at 37C for 30 min. A) X-ray autoradiography determined th e 1-nt cleaved product. B) Quantitative Analysis using electronic autoradiog raphy to measured the radioactivity. (InstantImager; Packard Instrument Co., Meriden, CT). The fraction of cleavage was calculated as the percent ra dioactivity present in the cleaved product as follows: % cleavage = [1-nt/(40-mer + 1-nt)]-100. Data are the mean SE of three different experiments. 73

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1234567 Control rabbit reticulocyte Fen-1wt Fen-1(1-115) Fen-1(78-192) Fen-1(154-268) Fen-1(231-345) Fen-1(269-380) +------+------+------+------+------+------+ 45 kDa 14 kDa 12-14 kDa 43 kDa NS MWFull length Fen-11 380 aa 1 115 aa 78 192 aa 154 268 aa 231 345 aa 269 380 aa A. Structure of the protein fragments B. Western blot Figure 4-6. Protein levels of in vitro transcription/translation of wild-type and fragments of Fen1. A) Depiction of protein fr agments of Fen-1. B) TNT synt hesis of Fen-1 wildtype and Fen-1 protein fragments. Proteins a nd protein fragments we re run on a 15% SDSPAGE followed by Western blot analysis Fen-1 wild-type protein and protein fragments were detected using mouse an ti-Fen-1antibody from Novus Biologicals. 74

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Fen-1wt Fen-1(1-115)Fen-1(78-192) 0 1 2.5 5 10 0 1 2.5 5 10 0 1 2.5 5 10APC peptide concentration ( g)APC (I-A,Y-A) APCwt APC (I-A,Y-A) APCwt APC (I-A,Y-A) APCwt Fen-1(154-268)Fen-1(231-345)Fen-1(269-380) 0 1 2.5 5 10 0 1 2.5 5 10 0 1 2.5 5 10APC peptide concentration ( g)APC (I-A,Y-A) APCwt APC (I-A,Y-A) APCwt APC (I-A,Y-A) APCwt Figure 4-7. Far-western analysis of the intera ction of APCwt and APC (I-A,Y-A) peptides and Fen-1 protein fragments. APC peptide wa s immobilized onto a PVDF membrane and incubated with either Fen-1 wild type or the indicated Fen1 protein fragment. Binding was detected through the use of a mouse anti-human Fen-1 antibody followed by chemiluminescence analysis. Fen-1 protein fragment 269-380 was the only fragment able to interact with APCwt. 75

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Fen-1(wt) Fen-1(K354A/R355A/K356A) Fen-1(K365A/K366A/K367A) 123 +--+--+ 43 kDa 45 kDa MW Fen-1wt Fen-1(K354A/R355A/K356A) mutant Fen-1(K365A/K366A/K367A) mutantA. Structure of wild-type and mutant Fen-1 proteinsLSSAKRKEPSTKKKAKT 13 5 0 358 363370380 aa LSSA AAA EPSTKKKAKT 13 5 0 358 363370380 aa LSSAKRKEPST KKKAKT 13 5 0 358 363370380 aaB. Western blot Figure 4-8. Protein levels of in vitro transcription/translation of wild-type and mutant Fen-1. A) Depiction of Fen-1 mutant proteins. B) We stern blot Analysis Depicting Result of TNT Synthesis of Fen-1 wild -type and mutant Proteins. Fen-1 wild-type and mutant proteins were synthesized using the TNT PCR Quick K it (Promega, Madison, WI). Synthesized proteins were run on a 10% SDS-PAGE and then transferred onto a PVDF membrane. Membrane was probed for Fen-1 wild-type and mutant proteins using a mouse anti-human Fen-1 antibody and the signal was detected using chemiluminescence analysis. 76

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Fen-1wt Fen-1 (K354A/R355A/K356A) Fen-1 (K365A/K366A/K367A) 0 2.5 5 10 15APC peptide concentration ( g)APC (I-A,Y-A) APCwt APC (I-A,Y-A) APCwt APC (I-A,Y-A) APCwt A. Far-Western blot0 2.5 5 10 15 0 2.5 5 10 15 B. Quantitative analysisTotal binding (%)0 80 60 40 20 120 100 APC(I-A,Y-A) ( g) APCwt ( g)0 2.5 5 10 15-----0 2.5 5 10 15-----0 2.5 5 10 15---------0 2.5 5 10 15-----0 2.5 5 10 15-----0 2.5 5 10 15Fen-1wtFen-1(K354A/ K355A/K356A) Fen-1(K365A/ K366A/K367A) Figure 4-9. Far-western analysis of the interaction of Fen-1 wild-type and mu tant proteins with APC. A) APCwt and mut peptides were immobilized on a PVDF membrane and incubated with Fen-1 wild-type and mutant proteins. Binding was detected using a mouse anti-human Fen-1 antibody followe d by chemiluminescence. B) Percent binding of APCwt and Fen-1 wild-type and mutant proteins. Percent binding was determined by using the density of APC and Fen-1 wild-type binding band as 100 % binding and comparing Fen-1 mutant proteins and APC-binding to the density of the APC and Fen-1 wild-type band [(Density of APCwt and Fen-1 mutant binding/Density of APCwt and Fen-1 wild-type) x 100 = Percent bound)]. Experiment was repeated twice. 77

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5 ng--+---+---+Bead Control----+ IP: Fen-1 WB: Fen-143 kDa A. Protocol B. Western blotFen-1wt Purified Fen-1 Reticulocyte control Fen1(K365A/K366A/K367A) 12345 Synthesis of protein by TNT Pull-down with anti-Fen-1 antibody Fen-1 bound with beads was used for endoand exonuclease assays 63-mer32P-labeled DNA 1 2 3 4 5 6 8 9 10 11 12----246----246 --64 2-75-nt products 5-nt flap 45-mer 23-mer 63-merPurified Fen-1 (nM) Reticulocyte control ( l) Fen-1wt ( l).2.1 -Fen-1(K365A/K266A/ K367A) ( l)-------1 2 3 4 5 6 8--36---3 ---76 ----1 0 -6 3 1-nt products *23-merC. Endonuclease activity63-mer D. Exonuclease activity Figure 4-10. Fen-1(K365A/K366A /K367A) mutant reta ins 5-flap endonuc lease and 5-3 exonuclease activity. A) Pull-down assay of wild-type and mutant Fen-1 proteins. Fen-1 wild type and Fen-1 (K365A/K366A/K36 7A) mutant protein were synthesized using the TNT PCR Quick Kit (Promega, Madison, WI) and then pulled down from the TNT reaction mixture using protein A sepharose beads and rabbit anti human Fen-1 antibody (Bethyl laborat ories, Montgomery, TX). Bead s were used in western blot analysis to detect presen ce of pulled-down Fen-1(wt) and Fen1(K365A/K366A/K367A) mutant pr otein. Proteins were det ected using anti-Fen-1 antibody (Novus Biologicals, Littleton, Co) and the chemiluminesence technique (GE Healthcare, Picastaway, NJ) B) Schematic of protocol used for endonuclease and exonuclease assay. C) Autoradiogram de picting endonuclease activity. Reaction mixtures in a 25 L final volume contained 2, 4, or 6 l of beads with Fen-1 wildtype or Fen-1(K365A/K366A /K367A) mutant protein. Pu rified Fen-1 wild-type protein at a concentration of 0.1 or 0.2 nM was used as a positive control. The mixture was incubated for 5 min on ice, and then 2.5 nM of the 32P-labeled flappedDNA substrate was added. The cleavag e reaction was carried out at 37oC for 30 min. X-ray film autoradiography determined the 5-nt cleaved product, and the electronic autoradiography measured the radioactivity (InstantImager; Packard Instrument Co., Meriden, CT). D) Autoradiogram depict ing exonuclease activity. Reaction was carried out as described in B with the following changes: 1. 3 or 6 l of beads with Fen-1 wild-type or Fen-1(K365A/K366A/K367A ) was used in the reaction mixture; 2. 10 nm of purified Fen-1 wild-type protei n was used as a postitive control; 3. A 32Plabeled nicked-DNA substrate was used; 4. X-ray film autoradiography determined the 1-nt cleaved product and electronic au toradiography measured the radioactivity. 78

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Figure 4-11. Imunohistochemical analysis of APC and Fen-1 co-localization in HCT-116APC(WT) and HCT-116-APC(KD) cells. Ce lls were treated with the indicated amount of MMS for 24 h and then stained for Fen-1 (green), APC (red), and nuclei (blue). Images were merged to determine co-localization. 79

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Alexa-594 (APC) Alexa-488 (Fen-1)Control MMSDAPI DAPI, Alexa-594 Alexa-488 merged Alexa-594, Alexa-488 mergedControl MMS Control MMS 5 h 15 h 24 hA.HCT-116-APC(WT)cells Alexa-594 (APC) Alexa-488 (Fen-1)Control MMSDAPI DAPI, Alexa-594 Alexa-488 merged Alexa-594, Alexa-488 mergedControl MMS Control MMS 5 h 15 h 24 hB.HCT-116-APC(KD)cells Figure 4-12. A time-course analysis of co-local ization of APC and Fen-1 in colon cancer cells treated with MMS. A) HCT-116-APC( WT) cells were treated with 500 M for the indicated amount of time. Cells were staine d for Fen-1 (green), APC (red), and nuclei (blue). Pictures were then merged for co-localization. B) HCT-116-APC(KD) cells. Cells were treated the same as HCT-116-APC(WT). 80

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Figure 4-13. A time-course analysis of co-local ization of APC and Fen-1 in LoVo cells treated with MMS. LoVo cells (truncated APC that lacks the DRI-domain) were treated with 500 M MMS for the indicated amount of time. Cells were stained for Fen-1 (green), APC (red), and nuclei (blue). Pictures we re then merged for co-localization. 81

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Time (h) HCT-116-APC(WT) HCT-116-APC(KD) MMS (500 M) +++++---------+++++ -++++-++++ Fen-10 10 15 25 5 0 10 15 25 5 APC +++++---------+++++ -++++-++++0 10 15 25 5 0 10 15 25 5A. Cytosolic fraction B. Nuclear fraction -Actin Figure 4-14.A time-course analysis of nuclear and cytosolic protein levels of APC and Fen-1 in MMS-treated colon cancer cells. HCT116-APC(WT) and HCT-116-APC(KD) cells were treated with 500 M MMS for the indicated amount of time, Nuclear and cyosolic fractions were prep ared and analyzed using west ern blot analys is. Fen-1 was detected using mouse anti-Fen-1 antibody (Novus Biologicals, Littleton, Co) while APC was detected using mouse anti-APC antibody (Millipore, CA). Signals were detected using the chemiluminescence techni que. A) Western blot analysis of APC and Fen-1 levels in the cytosolic frac tions of MMS treated HCT-116-APC(WT) and HCT-116-APC(KD) cells. -actin is shown as a loadi ng control. B) Western blot analysis of APC and Fen-1 levels in th e nuclear fractions of MMS treated HCT-116APC(WT) and HCT-116-APC(KD) cells. 82

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CHAPTER 5 DISCUSSION The Interaction of APC and DNA Polymerase and its Implications in BER In the present study the in teraction sites of poland APC involved in the APC and polinteraction were determined. Usi ng Y2H analysis with single am ino acid mutants of APC, it was found that only I1259 or Y1262 were necessary for the interaction of APC with pol. Furthermore, the mutation of either amino acid to alanine was enough to disrupt the interaction of APC with polsuggesting that the interaction of polmay be at the surface instead of in the groove of the APC protein. Additionally, the pres ent study indicates that APC interacts with a specific region of polBased on the Y2H analysis with pol-deletion construc ts and APC, it was determined that amino acids 80-120 of polwere critical for the interaction of polwith APC. Upon analysis of the crystal structure of poland examining the solvent accessibility of amino acids 80-120, possible amino acids involved in the interaction with APC were determined. Mutagenesis of Thr79, Lys81, and Arg83 to alan ine abolished the interaction between poland APC. Thus, amino acids Thr79, Lys81, and Arg83 we re identified as cri tical residues of polwhich were important for the interaction with AP C. Notably, these amino acids are located in the linker region of polwhich connects its lyase and pol ymerase domains (Beard and Wilson, 2006). The active site of the lyase domain of polis Lys72 which is closely located to the amino acids Thr79, Lys81, and Arg83 (Sriva stava et al., 1998). Due to this close proximity, it would be logical to hypothesize that the bindi ng of APC can interfere with pols deoxyribose phosphate (dRP)-lyase activity, and thus SP-BE R. In fact, studies conducted in dicate that this hypothesis is correct (Balusu et al., 2007). By reconstituting the SP-BER system in vitro these authors showed 83

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that SP-BER was blocked by APC in a dose-depende nt manner. Furthermore, they determined the mechanism by which APC is blocking SPBER by testing whether APC blocked poldRPlyase activity. The dRP-lyase activity of polhas been determined as the rate-limiting step in SP-BER. Removal of the 5dRP moiety is crucia l for the generation of the 5 phosphate required by DNA ligase to seal the nick and complete repair of the DNA (Beard and Wilson, 2006; Srivastava et al., 1998). Through the use of an in vitro dRP-lyase activity assay Balusu and colleagues were able to exam ine the effect of APC on pols dRP-lyase activity (Balusu et al., 2007). They found that an APC wt peptide was able to block pols dRP-lyase activity in a dose-dependent manner indicating that APC blocks SP-BER by modifying pols dRP-lyase activity. Moreover, they were also able to show that the strand-displacement synthesis of LPBER, which is blocked by APCwt, was unaffected with the polmutant (T79/K81/R83) protein. These results indicated that the phy sical interaction of APC with polis necessary to block its strand-displacement synthesis. Thus, it would ap pear that APC is a novel accessory protein in both SP and LP-BER. The Interaction of APC with Fen-1 and its Implications in BER Previous studies have show n that APC can block polstrand-displacement synthesis in LP-BER and interact with Fen-1 in vitro (Jaiswal et al., 2006; Na rayan et al., 2005). In the current study these findings have been extende d to examine whether APC and Fen-1 interact in vivo and if this interaction can block Fen-1 5enduonuclease and 5-3 exonuclease activities providing another method of inhi biting LP-BER. Furthermore, critical amino acid residues on Fen-1 and APC that participate in the Fen-1 and APC interaction have been determined. Lastly, immunohistochemical analysis and Western blot analysis of MMS-t reated cells have suggested a 84

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novel role for APC whereby it prevents Fen-1 from entering the nucleus after exposure to DNAalkylating agents. Through Y2H analysis it was determined that APCwt and Fen-1 could interact in vivo In light of this fact, it wa s decided to narrow down the interac tion site on APC for Fen-1. Since the interaction site on APC for polhad already been determined, it was thought that APC may interact with Fen-1 at the same site. Using Y2H analysis of single amino acid mutants of APC, it was found that the interaction site of APC for Fen-1 and polwas in the same domain. Only amino acids I1259 or Y1262 were necessary for the interaction of APC with Fen-1. Furthermore, the mutation of either amino acid to alanine wa s enough to disrupt APCs interaction with Fen-1 suggesting that the in teraction of Fen-1 may be at the surface instead of in the groove of the APC protein. Taken together, the fact that APC interacts with poland Fen-1 at the same site indicates that APC cannot interact with both proteins at the same time. This suggests that APC may have a preference for either protein or that poland Fen-1 may compete to bind with APC. Although how the differential interact ion of these proteins occurs in vivo is not clear. Fen-1 is the major protein involved in LP-BER. LP-BER can occur through two mechanisms: strand-displacement and Hit-and-Run synthesis. Fen-1 plays unique roles in each. In strand-displacement, Fen-1 cleaves the flap generated by pol, polor polmediated strand-displacement synthesis. In hit-and-run synthesis, Fen-1 excises a nucleotide at the 3-side of a nick to create a ga p to be filled by pol(Klungland and Lindahl, 199 7; Prasad et al., 2001; Qiu et al., 2001; Shibata and Nakamura, 2002) More specifically, th e two mechanisms proceed in the following manner. Once polarrives at an oxidi zed or reduced abasic site after APE incision, it proceeds to perform strand-displacem ent synthesis until Fen-1 binds and cleaves the displaced flap leaving a nick which is seal ed by DNA ligase. Altern atively, a hit and run 85

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mechanism of LP-BER can occur in which polincorporates 1-nt and the resulting flap is removed by Fen-1 5endonuclease activity. Then a series of sequential events occurs in which polbinds to the nick left behind by Fe n-1 endonuclease activity, extends the 3 -OH displacing the dRP moiety, fills the 1-nt gap, leaves a nic k, and dissociates from the DNA. Fen-1 then binds to the nicked DNA, removes 1-nt by its 5 3 exonuclease activity, creates a gap, and dissociates from the DNA. This cycle continues until DNA li gase seals the nick (Liu et al., 2005). As APC can interact with Fen1 and has already been shown to be involved in LP-BER, it was hypothesized that the in teraction of APC with Fen-1 may cause a blockage in Fen-1 catalytic activity, thus blocking LP-BER. Through the use of in vitro Fen-1 activity assays it was found that an APCwt peptide could block both Fe n-1 5endonuclease and 5 -3 exonucleas e cleavage abilities in a dose-dependent manner. Thus, APC is able to block both mechanisms of LP-BER. When APC blocks the 5endonucle ase cleavage activity of Fen1, it effectively blocks poldirected strand-displacement synthesis of LP -BER. However, when APC blocks the 5-3 exonuclease activity of Fen-1 it bl ocks hit and run synthesis of LP-BER. Thes e results combined with previous data show that APC can bloc k LP-BER in three ways: 1) By blocking poldirected strand-displacement synthesi s through APCs interaction with pol; 2) By blocking poldirected strand-displacement through APCs blockage of the 5 en donuclease activity of Fen-1; and 3) By blocking hit and run synthesis of LP-BER thr ough APCs blockage of the 5-3 exonuclease activity of Fen-1. Once it was found that APCs interaction with Fen-1 caused a block in its 5endonuclease and 5-3exonuclease activi ties, the next logical step was to identify the amino acids of Fen-1 necessary for its interaction with APC. Protein fra gments of Fen-1 were synthesized and used in a Far-western analysis with APC. Amino acids 346-380 of Fen-1 were found to interact with 86

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APC. Most of these amino acids lie within th e nuclear localization si gnal of Fen-1 suggesting that APC is interfering with nuc lear localization of Fen-1. I ndeed, two groups of amino acids essential for nuclear localiz ation (K354, R355, K356 and K365, K366, K367) lie within the region of amino acids 346-380 (Shen et al., 2005). Furthermore, three proteins which interact with Fen-1 have been found to interact at Fen-1s C-terminus between amino acids 353-380 (Guo et al., 2008; Sharma et al., 2005). Based on this knowledge two sets of amino acids Fen1(K354/R355/K356) and Fen-1(K365/ K366/K367) were chosen for further studies. These amino acids of Fen-1 are important in nuclear localization, and due to th e fact that they fell between amino acids 353-380 which have been shown to be the interaction site of Fen-1 with other proteins. Each amino acid was mutated to alanine and the mutant proteins were synthesized and used in a Far-western analysis with the APCwt peptide. Both mutants we re shown to reduce the interaction between APCwt peptide and Fe n-1. The Fen-1(K365A/K366A/K367A) mutant was able to reduce the interaction wi th APC to a greater extent. Th ese results suggested that APC does interact with the nuclear lo calization signal of Fen-1. More specifically, it suggests that APC interacts with the amino acids that have been shown to be essential for nuclear localization. To further characterize the interaction of APC with Fen-1, the synthesized Fen-1 (K365A/K366A/K367A) mutant was used in in vitro Fen-1 activity assays to determine if the Fen-1(K365A/K366A/K367A) mutant possess ed 5endonuclease and 5-3exonuclease activities. Surprisingly, the Fen-1(K365A/K366A /K367A) mutant retained both 5endonuclease activity and 5-3exonuclease act ivity. It was expected that the Fen-1(K365A/K366A/K367A) mutant protein would lose its 5endonuclease and 5-3exonuclease activities due to the fact that the interaction of APC blocks these activities. Since the Fen-1(K365A/K366A/K367A) mutant retains its endonuclease and exonuc lease activities, the physical interaction between Fen-1 and 87

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APC must be necessary for Fen-1 cleavage activity to be blocked. APC may cause a conformational change in Fen-1 upon binding that causes the active site of the endonuclease and exonuclease activities to become misaligned causing a blockage of Fen-1 endonuclease and exonuclease activities. Due to the fact that the interaction of Fen1 with APC appears to occur at the nuclear localization signal of Fen-1, it b ecame necessary to determine th e physiological significance of this interaction within the cell. Immunohistochemical analys is of HCT-116-APC(WT) cells treated with 500 M MMS for 24 h showed that most cells had an increased co-localization of APC and Fen-1 in the cytoplasm as well as a redu ction in nuclear Fen-1 as compared to control cells. This phenomenon was only seen in HCT-116-APC(WT) cells which contained wild-type APC. HCT-116-APC(KD) cells and LoVo cells were also treated with 500 M MMS, but failed to show a reduction in nuclear Fen-1 after 24 h of treatment. Since HCT-116-APC(KD) cells contain stably knocked down levels of APC and LoVo cells cont ain truncated APC which lacks the DRI-domain and therefore does not interact with Fen-1, these results i ndicate that wild-type APC must be present for the redu ction in nuclear Fen-1 to occur. Furthermore, this reduction occurs in response to DNA-alkylation damage. To further understand how this reduction in nuclear Fen-1 occurs after treatment with MMS for 24 h, a time course experiment was conducted where the aforementioned cell lines we re treated with MMS for 5, 15, or 24 h. Once again the reduction in levels of nuclear Fen-1 and increased co-localization between APC and Fen-1 were only seen in HCT-116-APC(WT) cells. After 15 h of treatment with 500 M MMS about half of the HCT-116-APC(WT) cells exhi bited a reduction in nuclear Fen-1 and by 24 h most of the HCT-116-APC(WT) cells exhibited a loss of nuclear Fen-1. Cells treated with MMS for 5 h did not exhibit a loss of nuclear Fen-1, bu t they did exhibit increased co-localization of 88

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APC and Fen-1 in both the cytoplasm and the nucleus. In contrast these observations were not seen in HCT-116(KD) cells or LoVo cells that received the same treatment. Furthermore, the increased co-localization of APC and Fen-1 wa s not apparent in HCT-116-APC(WT) cells which did not receive the MMS treatment. These result s were further confirmed through Western blot analysis. HCT-116-APC(WT) cells and HCT-116-APC (KD) cells were treated with MMS for 5, 10, 15, and 25 h and the nuclear and cytosolic fractio ns were collected. Thes e fractions were then used in Western blot analysis to determine the levels of APC and Fen-1 in the nucleus and the cytoplasm in MMS-treated HCT-116-APC(WT) a nd HCT-116-APC(KD) cells. An increase in cytoplasmic APC was seen in HCT-116-APC(WT) cells with the maximum increase being at 15 h after MMS treatment as compared to control HCT-116-APC(WT) cells. Interestingly, levels of nuclear Fen-1 were decreased in HCT-116-APC(WT) cells in response to MMS treatment with the maximum decrease seen at 15 h suggesting that an increase in cytoplasmic APC may lead to a reduction in nuclear Fen-1. As expected, this phenomenon was not seen in the cytoplasmic or nuclear fractions of MMS-treated HCT-116-APC(KD) cells. Levels of APC were not detected in the cytosolic or nuclear fractions of MMS treated HCT-116-APC(KD) cells, while Fen-1 levels showed no significant change in both the cytoso lic and nuclear fractions. These results support the hypothesis that APC interacts with Fen-1 at its nuclear localization signal and blocks the translocation of Fen-1 to the nucleus. Interest ingly, the highest level of nuclear APC in HCT116-APC(WT) cells was seen at 25 h after MMS treatment. This correlated with a modest increase in the levels of nuclear Fen-1 providi ng further support for the hypothesis that APC can interact with Fen-1 and block its catalytic activity. Combined, these results suggest another m echanism of APC-mediated block of BER by retaining Fen-1 in the cytoplas m in response to DNA alkylation da mage. The data suggests that 89

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APC becomes upregulated in response to MMS tr eatment. Over the course of treatment, APC interacts with Fen-1 at its nuclear localizati on signal in the cytoplasm and prevents it from entering the nucleus causing a loss of Fen-1 in the nucleus due to the fact that the Fen-1 protein cannot be replenished. In summation, APC can bloc k LP-BER through its in teraction with Fen-1 via two mechanisms. First, APC can block LP-B ER by binding to Fen-1 and causing a block in both Fen-1 5endonuclease and 5-3exonuclease cleavage activities. Sec ondly, in response to DNA damage, APC can block LP-BER by binding to Fen-1 at its nuclear localization signal and prevent it from replenishing nuclear Fen-1. Whethe r APC interacts with newly synthesized Fen-1 and whether post-translational modifications in APC and/or Fen-1 after DNA-alkylation damage causes cytoplasmic co-localization of APC and Fen-1 is not yet known. Conclusions and Future Implications Mutations in the APC gene are among the earliest events in colorectal carcinogenesis. The blockage of BER by APC may be viewed as a pa radox as APC is generally viewed as a tumor suppressor. The blockage of BER by APC could serve as a tumor suppressor if the blockage of BER leads to apoptosis as a result of the accu mulation of DNA damage. Indeed, studies have found that treatment of human colon cancer cel ls and mouse embryonic fibroblast cells with MMS enhanced the levels of APC and blocked BER, resulting in increased sensitivity and apoptosis of cells harboring damaged DNA (Kundu et al., 2007b; Narayan et al., 2005). Furthermore, it has been shown that HCT-116APC(WT) cells treated with MMS exhibited decreased colony formation in a clonogenic assa y when treated with MMS compared to HCT116-APC(KD) cells treated with MMS indicating that increase d levels of APC after MMS treatment blocks BER and decreases cell grow th (Kundu et al., 2007b). Th ese results indicate that APC plays a role as a tumor suppresso r by removing cells containing damaged DNA. Alternatively, studies have shown that cigare tte smoke condensate induces APC levels in 90

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91 spontaneously immortalized normal human breas t epithelial cells, bl ocks BER, and causes transformation of these cells (Kundu et al., 2007a). These findings point to a role of APC in carcinogenesis. In conclusion, the findings of the present study indicate that APC can block LP-BER by interacting with Fen-1 through two different mech anisms: direct interact ion with Fen-1 causing blockage of its endonuclease and exonuclease activities and by bi nding to Fen-1 in response to DNA damage and preventing Fen-1 from entering the nucleus to replenish nuclear Fen-1. Furthermore, it was discovered that the linker region of polis responsible for the interaction with APC indicating that this region of polmay play a role in the complex regulation of the BER process in vivo These proteins, including APC, may be involved in specific subpathways of BER that could be trigge red by unknown signaling. Studies ch aracterizing these subpathways and the signaling processes that control them will serve to shed some li ght on the regulation of BER in vivo and the role of APC in this regulation. The role of APC in the blockage of LP-BER may provide clinical implications in the future. Since the DRI-domain is located toward the N-terminal region of the APC protein, which is spared by the MCR region, both wild-type and mutant APC proteins (containing the DRIdomain) may block DNA damage-induced LP-BER. Th erefore, if the chemopreventative agents that produce DNA damage can increase APC levels in the target cell, then the decreased LP-BER and increased DNA damage may induce cell deat h. Thus, the link between APC and DNA repair may prove important in the development of chemotherapeutic drugs in the future.

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Faux, M.C., Ross, J.L., Meeker, C., Johns, T., Ji H., Simpson, R.J., Layton, M.J., and Burgess, A.W. (2004). Restoration of full-length adenom atous polyposis coli (APC) protein in a colon cancer cell line enhances cell adhesion. J Cell Sci 117 427-439. Fodde, R., Kuipers, J., Rosenberg, C., Smits, R., Kielman, M., Gaspar, C., van Es, J.H., Breukel, C., Wiegant, J., Giles, R.H., and Clevers, H. (2001). Mutations in the APC tumour suppressor gene cause chromosomal instability. Nat Cell Biol 3, 433-438. Frank, G., Qiu, J., Zheng, L., and Shen, B. (2001). Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PC NA) is independent of its in vitro interaction via a consensus PCNA binding region. J Biol Chem 276, 36295-36302. Gary, R., Park, M.S., Nolan, J.P., Cornelius, H.L., Kozyreva, O.G., Tran, H.T., Lobachev, K.S., Resnick, M.A., and Gordenin, D.A. (1999). A nove l role in DNA metabolism for the binding of Fen1/Rad27 to PCNA and implications for genetic risk. Mol Cell Biol 19 5373-5382. Gerson, S.L. (2004). MGMT: its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 4, 296-307. Gorbunova, V., and Levy, A.A. (1997). Non-homol ogous DNA end joining in plant cells is associated with deletions and fille r DNA insertions. Nucleic Acids Res 25, 4650-4657. Green, R.A., and Kaplan, K.B. (2003). Chromosome instability in colorectal tumor cells is associated with defects in mi crotubule plus-end attachments ca used by a dominant mutation in APC. J Cell Biol 163, 949-961. Green, R.A., Wollman, R., and Kaplan, K.B. (2005). APC and EB1 function together in mitosis to regulate spindle dynamics and ch romosome alignment. Mol Biol Cell 16 4609-4622. Grote, H.J., Schmiemann, V., Kiel, S., Bocking, A., Kappes, R., Gabbert, H.E., and Sarbia, M. (2004). Aberrant methylation of the adenomat ous polyposis coli promoter 1A in bronchial aspirates from patients with su spected lung cancer. Int J Cancer 110, 751-755. Guo, Z., Chavez, V., Singh, P., Finger, L.D., Hang, H., Hegde, M.L., and Shen, B. (2008). Comprehensive mapping of the C-terminus of fl ap endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication and repa ir pathways. J Mol Biol 377, 679-690. Hamada, F., and Bienz, M. ( 2002). A Drosophila APC tumour suppressor homologue functions in cellular adhesion. Nat Cell Biol 4, 208-213. Hamada, F., and Bienz, M. (2004). The APC tu mor suppressor binds to C-terminal binding protein to divert nuclear beta -catenin from TCF. Dev Cell 7, 677-685. Hamid, S., and Eckert, K.A. (2005). Effect of DNA polymerase beta loop variants on discrimination of O6-methyldeoxyguanosine modifi cation present in the nucleotide versus template substrate. Biochemistry 44, 10378-10387. 95

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107 BIOGRAPHICAL SKETCH Melissa Liz Armas was born in Miami, Florida, to Cuban parents. She is the only daughter of Mayra and Eser Armas. Since an early age, she was interested in Science. Melissa attended Southwest Miami Senior High School, where she was an honors student and was active in the Phi Beta Chi Science club. She was also activ e in the Foreign Language club and the Future Business Leaders of America. After graduating high school, Me lissa attended the University of South Florida, where she received a B.S. in biology. She was also part of the Honors Program. Melissa was accepted into the Ronald E. McNair Postbacculaurete Achievement Program. As a McNair Scholar, Melissa received her first research experien ce. Her research project was on Brain cancer (Glioma) cells. She was able to present her re search at McNair conferences and the Honors research symposia. After graduating from the University of S outh Florida, Melissa enrolled into the IDP program at the University of Florida to pursue her doctorate. She pursued her doctoral research on Adenomatous Polyposis Coli (APC) and its in teraction with Base Excision Repair proteins: Its role in co lorectal carcinogenesis in the laboratory of Satya Narayan. Melissa has presented her research in departmental seminars and has had two abstracts presented at national meetings. Melissa plans to pursue a ma sters in public health. By combining the knowledge from her doctorate and her masters, Melissa plans to focus on Health Policy and its relation to minority populations.


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