Citation
Building an Episomal Model of Aging in Saccharomyces cerevesiae

Material Information

Title:
Building an Episomal Model of Aging in Saccharomyces cerevesiae
Creator:
FALCON, ALARIC ANTONIO ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Cellular senescence ( jstor )
Daughter cells ( jstor )
DNA ( jstor )
Life span ( jstor )
Magnetism ( jstor )
Monomers ( jstor )
Mother cells ( jstor )
Plasmids ( jstor )
Ribosomal DNA ( jstor )
Yeasts ( jstor )

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Alaric Antonio Falcon. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
4/30/2004
Resource Identifier:
72844070 ( OCLC )

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Full Text











BUILDING AN EPISOMAL MODEL OF AGING IN Saccharomyces cerevisiae


By

ALARIC ANTONIO FALCON















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

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Alaric Antonio Falc6n



































This document is dedicated to Peri A. Tong, Manuel A. Falc6n, and Beverly L. Metcalfe
for their unwavering support.
















ACKNOWLEDGMENT S

I thank my mentor, John P. Aris, and my committee (William A. Dunn, Thomas C.

Rowe, and Brian Burke) for helping me become a scientist.




















TABLE OF CONTENTS
Page

ACKNOWLEDGMENT S ........._._._..... .___ ..............iv.....

LI ST OF T ABLE S .............. ..............viii....


LI ST OF FIGURE S ........._._._..... .___ .............._ ix...

AB S TRAC T .........._.._.._ ..............xi..._.... ....


CHAPTER

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


Sirdp, rDNA, and Aging .............. ......._ .. .............. 1..
Extrachromosomal rDNA Circles are Discovered ......._.__ ........__ .............2
Components of the rDNA .........._.._...... .............. 3...
Foblp and its Role in ERC Production .........._.__..... .__ .. ....._._.......
ARS of the rDNA .........._..... ........_ ..............6....
Asymmetric Inheritance of ERCs .........._.._........_....._ ............
Summary .........._.__..... .__ ..............7.....


2 DEFINING THE LINK BETWEEN EPIS OMES AND AGING .............. ... ............9


Roles of Different Cis-Acting Plasmid Sequences in Reduction of Yeast Replicative
Life Span ................. .... ......... ............ .... ... ........... .......1
Plasmid Inheritance Correlates with Reduction in Yeast Life Span ................... ...... 17
Plasmids Do Not Significantly Increase ERC Levels ................. ................. ....20
Plasmid Accumulation Correlates with Reduction in Life Span .............. .... .......... 22
Terminal Cell M orphology ........._...... ........ .._.._ ..... ...._.._....... ...........2
Do Functional rDNA Transcriptional Units Play a Role in Reduction in Life Span?31
Summary ............ _...... ._ .............. 3 3...


3 TWO MICRON CIRCLE: A NATURALLY OCCURRING EPISOME' S ROLE IN
AGING ................. ................. 3......... 5.....


New Method for Removal of Two Micron Circle ................. ................ ...._ 36
Two Micron Circle Does Not Reduce Life Span ................ ... .............. ........38
Two Micron Circle Does Not Accumulate in Old Cells ................. ................. ..39
Summary ................. ................. 41.............











4 A CELL'S LIMITED RESOURCES AND PLASMID COMPETITION .................42


Smllp, Meclp, RNR, and Rad53p Pathway ................ ..............42. ...........
SML 1 Deletions Do Not Increase Life Span. ........._._........___ .........._...43
Old Cells Do Not have an Increased Sensitivity to Hydroxyurea. ........._...........45
Double Strand Breaks Do Not Increase in Old Cells .........._.... ........__........48
Phosphorylation of Rad53p Does Not Increase in Old Cells. ............. ...... ........ 49
ERC Competition with a CEN Plasmid Throughout Yeast Life Span ................... ... 50
Mitotic Stabilities in the Presence of ERCs ......___ ..... ...__ ........_..... 52
Plasmid Accumulation in sir2A and foblA Strains ................ ................ ...._ 55
Summary ................. ................. 56.............

5 CHROMATIN SILENCING AND EPISOME FORMATION ................ .............. 58


ACS2 and ACS1 is Required for Normal Life Span ................ ................ ...._ 59
ACS2A Increases ERC Production ................ ..............61. ..............
Summary ................ ..............62. ...............

6 LOOKING AT POSSIBLE MECHANISM OF CELLULAR AGING ................... ..64

YCA1 and Apoptosis in Yeast .............. ... .......... ...............64....
Shu Gene Family and Mutation Suppression in Aging ................. ................. ...65
Summary ................. ................. 67.............

7 DI SCU SSION ................. ................. 69.............


Why Do ARS Plasmids Accumulate in Mother Cells? ........... .. .. .........___......70
Why Do Budding Yeast Exhibit a Mother Cell Plasmid Segregation Bias?............. 71
Why Do ARS1~ Plasmids Bring About Cellular Senescence More Rapidly than Do
ER C s? ............ .... ........._ ........_ .. .. .............7
Do Cis-acting Sequences that Counteract Mother Cell Segregation Bias Suppress
Reduction in Life Span by ARS1~ Plasmids?.............. ................72
Do 2 Micron Circles Reduce Life Span? ......____ .... .. .__ .......__........7
Why Do 2 Micron Origin Plasmids Reduce Life Span? .............. ..............___....72
Why Do 2 Micron Origin Plasmids Have an Intermediate Effect on Life Span?......73
Why Does Transformation with pJPAll4 Lead to 2 Micron Circle Loss?...._........73
By What Mechanism(s) Do Plasmids, and by Implication ERC s, Reduce Life Span
in Y east? ........._.._.. ... ... ..._._ ..... .. .._ ..... .. .. ........7
Why Are There Less Plasmid Accumulation in Strains that Produce More ERCs?.. 75
Is There Episomal Aging in Metazoans? ....__ ......_____ .... .....__........7

8 MATERIALS AND METHODS ............_...... .__ .....___..........7

Yeast Strains and Plasmids ............ .....__ ..............77..
M itotic Stability .............. .. ........... ..............79.......
Replicative Life Span Determinations.............. .............79
Southern Blot Analysis and Quantitation ....__ ......_____ ..... .....__.........8











Magnetic Cell Sorting.............. ................ 8 1
Budscar Histograms ........._._ ......._. .............. 82...
rDNA Recombination Assay .........._._ ...._.._ .. .............. 82...

APPENDIX


A STRAIN: W303AR5+pJPAl l3 MEDIA: SD aHLW .................... .............. 84

B STRAIN: W303AR5+pJPAll6 MEDIA: SD aHLW.........._.._.. ........_.._........ 85

C STRAIN: W303AR5+pJPAl38 MEDIA: SD aHLW .........._.._.. ........_.._........ 86

D STRAIN: yAF6 MEDIA: SD aHLW .............. .................... 87

E STRAIN: W303AR5+pJPAl33 MEDIA: SD aHWu.............. .... .............. 88

F STRAIN: W303AR5+pJPAl36 MEDIA: SD aHWu.............. .................. 89

G STRAIN: W303AR5+pJPAl48 MEDIA: SD aHWu.............. ..................90

H STRAIN: yAF5 MEDIA: SD aHWu.............. ..............91...

I STRAIN: W303R5+pAF32 MEDIA: YPD.............. ...................92

J STRAIN: FOB1A+pAF32 MEDIA: YPD ....__ ......_____ ..... ......._.......93

K STRAIN: SIR2A+pAF32 MEDIA: YPD ........................... .......94

L PLASMIDS USED ............ _...... ._ ..............95....

M STRAINS USED ............ _...... ._ ..............96....

LIST OF REFERENCES.............. ...............99

BIOGRAPHICAL SKETCH ............_...... .__ ............._ 105...


















LIST OF TABLES

Table pag

2-1. Plasmids used in this study. ................ ................. 13......... ..

2-2. Life span data summary.............. ................ 16

6-1. P-values of the SHU deletion life spans. .........._._...... .__ ....._._........6

L-1. The plasmids used throughout this dissertation.............. .............. 95

M-1. The strains used throughout this dissertation. ................ ......... ................ 96


















LIST OF FIGURES


Finure pag

1-1. The pseudoERC strategy ............ ...... ._ ..............3...

1-2. The rDNA repeat. .........._...._ ..............4........_ .....

1-3. Fob 1 mediated expansion of the rDNA. .........._...._ .........._....5_....._.._..

1-4. ERC Formation ............ ..... ._ ..............6....

2-1. Life span analysis of plasmid-transformed yeast. ................ ................ ...._ 15

2-2. Plasmid inheritance studies ........._..... .............. 19.__.. .....

2-3. Extrachromosomal rDNA circle (ERC) formation in yeast transformants ............22

2-4. Plasmid DNA and extrachromosomal rDNA circle (ERC) levels in young and old
cell s ....... ..... ................. 24..............

2-5. Plasmid DNA and extrachromosomal rDNA circle (ERC) levels in young and old
cell s ....... ..... ................. 28..............

2-6. Terminal morphology of senescent cells.............. ................. 30

2-7. Life span analysis of yeast transformed with plasmids containing rDNA repeats.. 32

3-1. Southern blot analysis of pJPAll14 transformants ................ ................. ...._37


3-2. Life span analysis of cir+ and cir0 yeast ................ ..............39. .......... .

3-3. Two micron plasmid levels in young and old cells. ................ ... .............. .....40

4-1. Meclp, Rad53p, Smllp, and RNR pathway (94)............_._. .......___ ........._43

4-2. Life span of SM~L1 deletions ................. ......... ........ ...........4

4-3. Hydroxyurea sensitivity of young and old cells from 0 mM to 200 mM ............... 46

4-4. Hydroxyurea sensitivity of young and old cells from 0 mM to 50 mM. ................ 47











4-5. Southern of DSB in yAF6 (WT), W1488-4C (WT), smllA, meclAsml~A, and
rad53 Asml lA.. ................ ................. 49.............

4-6. Rad53p phosphorylation in young and old cells.............. ................ 50

4-7. Life span of W303R5 (WT), sir2A, and foblA during the CEN loss experiment...5 1

4-8. The age which WT, sir2A, and foblA lose plasmid.............. ................ 52

4-9. Mitotic stabilities of pAF3 1 and pAF32 ................ ................. 53...........

4-10. Mitotic stabilities of pJPAl33 and pJPAl36. ................ ......... ................ 54

4-11. Southern of showing plasmid competition phenomenon.. ................ ................. 55

4-12. Quantitation of plasmid competition Southern ....._____ .......___ ............. 56

5-1. The acetylation of histones and its affect on ERC production and life span............ 59

5-2. Life span of ACS deletions. ........._._. ....___......_. ..........6

5-3. Sorts of young and old ACS deletion strains. ....._._._ ..... ..__. .................62

6-1. Life span of ycalA. ........._._ ......._. ..............65...

6-2. SHU genes role in life span. .........._.__.....__ ......_ .........6
















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

BUILDING AN EPISOMAL MODEL OF AGING IN Saccharomyces cerevisiae

By

Alaric Antonio Falcon

May, 2004

Chair: John P. Aris
Major Department: Anatomy and Cell Biology

Aging in Saccharomyces cerevisiae is under the control of multiple pathways. The

production and accumulation of extrachromosomal rDNA circles (ERC s) is one pathway

that has been proposed to bring about aging in yeast. To test this proposal, we developed

a plasmid-based model system to study the role of DNA episomes in reduction of yeast

life span. Recombinant plasmids containing different replication origins, cis-acting

partitioning elements, and selectable marker genes were constructed and analyzed for

their effects on yeast replicative life span. Plasmids containing the ARS1 replication

origin reduce life span to the greatest extent of the plasmids analyzed. This reduction in

life span is partially suppressed by a CEN4 centromeric element on ARS1~ plasmids.

Plasmids containing a replication origin from the endogenous yeast 2 micron circle also

reduce life span, but to a lesser extent than ARS1 plasmids. Consistent with this, ARS1

and 2 micron origin plasmids accumulate in ~7-generation-old cells, but ARS1/CEN4

plasmids do not. Importantly, ARS1~ plasmids accumulate to higher levels in old cells

than 2 micron origin plasmids, suggesting a correlation between plasmid accumulation










and life span reduction. Reduction in life span is not an indirect effect of increased ERC

levels, nor the result of stochastic cessation of growth. The presence of a fully functional

9.1 kb rDNA repeat on plasmids is not required for, and does not augment, reduction in

life span. These findings support the view that accumulation of DNA episomes,

including ERC s, cause cell senescence in yeast.

The endogenous 2 micron circle is a naturally occurring episomal DNA. Loss of

the 2 micron circle can be facilitated with the transformation of an ARS containing

plasmid. Since 2 micron circles are episomes, and episomes can cause aging,

experiments were complete to show that it does not accumulate in old cells and does not

cause agmng.

In strains that contain more ERCs, ARS plasmids do not accumulate as much.

There is an episomal competition phenomenon. While it is not known what the episomes

are competing for, it can be demonstrated that as the number of different episomes

increase the rate of accumulation for each episome decreases.















CHAPTER 1
BACKGROUND AND SIGNIFICANCE

Saccharomyces cerevisiae is a single-ce Iled, budding yeast. During mitosis,

budding yeast divide asymmetrically. This is different from fission yeast where the two

cells produced by mitosis are indistinguishable. With budding yeast, the new smaller cell

(the daughter cell) emerges from the older, larger cell (the mother cell) (1-4). Because

the mother cell can be distinguished from its daughters, a mother can be followed

throughout all of its divisions. As mothers age, they become enlarged, their cell cycle

slows, and they become sterile (2,5). Daughters can be physically separated from their

mothers with a microdisection microscope. Physical separation is necessary, because it is

difficult to follow a mother cell through all of its divisions if it is obscured by daughter

cells and daughters of daughter cells. By removing the daughters from mothers, and

simultaneously tallying the number of divisions a mother completes, the cell's replicative

life span can be determined. The replicative life span is defined as the number of

divisions a mother cell completes (6). With the ability to conduct replicative life span

experiments and the ease of genetic manipulation, Saccharomyces cerevisiae is an

excellent model organism for aging studies.

Sir2p, rDNA, and Aging

Long and short lived aging mutants have been isolated in Saccharomyces

cerevisiae (7-10). One of the first such mutants identified was SIR4-42, a long lived

mutant that resulted in the localization of the SIR complex to the nucleolus (3,8). The

silent information regulator (SIR) complex is involved in the silencing of chromatin at









the telomeres, mating type loci, and rDNA (2,11,12). The rDNA serves as a nucleolar

organizing region in eukaryotic cells, and is the site of transcription of pre-rRNA (11). In

addition to the findings that implicated the rDNA in life span, the nucleolus was found to

be enlarged and fragmented in old yeast cells (2,3). This was consistent with the notion

that the rDNA played a role in life span determination. More recently, the silencing

protein Sirdp was found to be a nucleolar protein specifically involved in silencing at the

rDNA locus (3). Loss of function sir2 mutations reduce life span (3,12), whereas SIR2

overexpression extends life span (13). Sirdp is now known to function as a histone

deactylase that plays a central role in modulating chromatin structure (13,14). It has been

tied to the extension of life span in metazoan organisms (15), as well as being linked to

the caloric restriction model of aging (16).

Extrachromosomal rDNA Circles are Discovered

Based on SIR4-42 and other findings, Sinclair and Guarente in 1997 showed that

old mother cells accumulated extrachromosomal rDNA circles (ERCs). This was proven

by the use of 2D chloroquine gels and by Southern blots for rDNA. 2D chloroquine gels

are used to look for closed circular DNA molecules (17-19). Old cell undigested DNA

on 2D chloroquine gels showed rDNA episomes. The old cells had rDNA episomes and

young cells did not (20). To test the role of ERCs in yeast mother cell aging, replicative

life spans were conducted on cells that had been given pseudoERCs. PseudoERCs are

induced by using a plasmid with a partial rDNA repeat flanked by loxP sites and a

plasmid with Cre recombinase under the control of the galactose promoter. On adding

galactose, the galactose promoter allows the expression of Cre recombinase (21). Cre

recombinase then recombines the sequence between the loxP sites out of the plasmid

(22). This creates the pseudoERC, a partial rDNA repeat with a selectable marker and no










extraneous segregation or replication mechanisms (Figure 1-1). These pseudoERCs

cause an earlier onset of senescence (20). More specifically, the cell that had the

induction of pseudoERCs had a lower replicative life span (20). This suggested that

ERCs are a cause of aging, and are not produced as an effect of aging. This experiment is

the central proof of the ERC mediated aging model. The finding that ERCs could cause

aging was a completely novel aging mechanism.













producesDE Crep- rcmiaeC reomediatesrecobnto atr th lxPsie
resultingin the exision ofthe ASCNadteceaino suoR



Compoentsof th rDN





The ribosomal DNA (rDNA) is present on chromosome XII in a single linear array

of between 100 and 200 head-to-tail repeats (2,3,11,23). Each 9. 1 kb rDNA repeat is

responsible for producing the 5S and 35S pre-rRNA (Figure 1-2) (7,24) These RNAs

are later processed and packaged with proteins to form a ribosome (24,25). One half of

rDNA repeats are usually silent and not actively transcribing the rRNA (11,26). This

excess capacity is to ensure that the vital function of protein synthesis will not be

hindered by the lack of rRNA.






















Figure 1-2.The rDNA repeat. 5S and 35S pre-rRNA are both transcribed in the rDNA.
The 35S is later processed into 18S, 5.8S, and 25S rRNA. Within the NTSs
are the RFB and ARS-rD (both have been shown to be essential for ERC
formation and replication, respectively).

Foblp and its Role in ERC Production

The protein Foblp is required for the replication fork block (RFB) in the

non-transcribed spacer 1 (NTS1) (23,27). It is implicated in the formation of ERCs (7).

The RFB blocks one of the replication forks within the replication bubble (23). This

makes replication unidirectional, in the direction of 35S pre-rRNA transcription (23).

The RFB has been observed in yeast, frog, mouse, human, and plant (28-32). The

ultimate function of the RFB site in yeast is to allow the cell to expand and contract the

number of repeats in the rDNA array by homologous recombination (23) (Figure 1-3).

Because recombination is initiated at a DNA double stranded break (DSB), a crossover

may occur within a sister chromatid by the formation of a Holliday structure (23).

Conversely, recombination can occur upstream, resulting in the loss of a repeat (23). An

apparently unintended consequence of RFB function is the increased production of ERCs

(Figure 1-4) (7). Holliday structures within the rDNA occur 3.6 times per cell cycle (33),

indicating rampant recombination at the locus. In FOB1 deleted strains, there is reduced

recombination at the rDNA locus, because there is no RFB at the RFB site (34). This





















































Figure 1-3.Fob1 mediated expansion of the rDNA. (A) FoblIp acts at each RFB site in
rDNA. (B) Replication begins at ARS-rD and two replication forks travel in
opposite directions. (C) The replication fork traveling in the opposite
direction of 35S transcription is stopped at RFB site. The other replication
fork continues. A double stranded break can occur at the RFB site. (D) A
Holliday structure forms and homologous recombination with the sister
chromatid can repair the break. (E) The closest replication bubble catches up
to the recombination site creating two separate strands of DNA, one of which
has an additional repeat.


~s-~ ~-r


I~


-I1~L


__ __ __ __ _~ _~ _


-~L ~68r


reflects foblA' s reduced formation of ERCs and longer life span (7). This further

implicates ERCs in the aging process.


----


-r-~--~


C ~._~-gL~e. rqs8, ~L ~dL~


~

J~k~


~91P


~'118
~~B








A B3








O, O

Figure 1-4.ERC Formation (A) An ERC can be formed by homologous recombination
and the looping out a circular DNA. (B) They can also be formed by
recombination of the free end of DNA, the product of FoblIp RFB (27).
ARS of the rDNA

Within every rDNA repeat, there is an autonomously replicating sequence (ARS)

or origin of replication (3 5,36). ARSs are AT rich sequences of DNA, to which the

origin recognition complex (ORC) binds (37,38). The ORC complex of proteins is

essential for the initiation of DNA replication (37,3 8). The ARS-rD's (rDNA' s ARS)

biological function is as a site for the initiation of DNA replication within the rDNA

repeat. It is necessary because the repeat locus consists of one to two hundred
head-to-tail 9.1 kb repeats (approximately 1,500 kb in length) (39). Normally, ARSs are

spaced approximately 40 kb apart throughout the yeast genome (37,39,40). By putting an
ARS in every repeat, replication of the genome through this lengthy region can occur

more efficiently than with an ARS at either end of the locus (39). The ARS will also

allow episomal rDNA, such as ERCs, to replicate (20,35). The ARS-rD is considered a

"weak" ARS. That is, any given ARS-rD fires less than once per cell cycle (35,36,39).

Because ARS-rD occurs once in every repeat, every third ARS can fire and still replicate

the locus effectively. The distance between firing ARSs is approximately 30 kb. The









activity of the ARS-rD has been linked to transcriptionally active 3 5S genes (which were

in turn linked to nonsilent euchromatic regions of the locus) (41,42).

Asymmetric Inheritance of ERCs

A phenomenon associated with ERCs is their asymmetric segregation. There is a

natural tendency for the ERCs and ARS plasmids to stay within the mother cell during

cell division (2,3,43). This was demonstrated by pedigree analysis, a technique that

follows the segregation of a non-Mendelian trait through mitosis (20). Mother cells have

a bias to retain the plasmids and not pass them on to their daughters (43). Although this

phenomenon was discovered in 1983, little is known about the mechanism that retains the

plasmids in the mother cells. With ERCs being excised from the genome, replicated by

their endogenous ARS, and segregated preferentially to the mother; there is a massive

accumulation of the episomes in older cells. This is the model of ERC mediated aging

(20). Although the amount of ERCs in very old cells is not known, the number estimated

to be in cells after 15 generations is 500 to 1000 ERCs (20). This accumulation is

thought to be behind the mechanism of ERC mediated aging (20).

Summary

A maj or tenet of the ERC mediated model for replicative aging in Saccharomyces

cerevisiae is that ERCs are nothing more than episomal DNA molecules with an ARS

(20). Evidence for this comes from the observation that a yeast shuttle vector, containing

only an ARS, reduced replicative life span (compared to a control plasmid containing an

ARS and a centromeric (CEN) element) (20). CEN plasmids are maintained at low copy

number and segregate with high fidelity to daughter cells just like chromosomes (44).

ARS plasmids attain a high copy number and show a bias toward retention in mother

cells during mitosis (similar to ERCs) (43). The fact that the ARS plasmid can shorten









yeast mother cell life span suggests that the rDNA sequence per se does not contribute to

ERC mediated aging, and that potentially any extrachromosomal DNA able to replicate

may reduce life span (20).















CHAPTER 2
DEFINING THE LINK BETWEEN EPISOMES AND AGING

The yeast Saccharomyces cerevisiae has proved to be a valuable model organism

for investigating mechanisms of cellular aging (45-47). Central to the biology of aging in

S. cerevisiae is an asymmetric cell division process that gives rise to mother and daughter

cells with different characteristics. Mother cells have a limited capacity to produce

daughter cells, and the decline in this capacity with each generation is referred to as

replicative aging. The limited replicative potential of yeast mother cells has been

recognized since the 1950s (48). Pioneering studies in the Jazwinski and Guarente

laboratories postulated the existence of a senescence factor/substance that accumulates in

mother cells and is transmissible to daughters (49,50). Work in the Guarente lab

identified a heritable "age" locus that regulates yeast life span (51). More recent studies

have made clear that allelic variation at single genetic loci can markedly affect yeast life

span, including extension of life span. This indicates that a process as complex as

cellular aging is controlled by a hierarchical regulatory system. Like in other model

organisms, such as D. melan2oga~ster and C. elegan2s, mutations that influence yeast life

span have been found to exert their effects through different physiological and genetic

pathways, including those that participate in caloric restriction, gene silencing, genomic

stability, growth regulation, mitochondrial function, and stress response (45-47,52,53).

Replicative aging is undoubtedly a complex process, even in a eukaryote as

simple as S. cerevisiae. Different hypotheses have been proposed to explain yeast

replicative aging. One hypothesis proposed by Sinclair and Guarente (54) posits that










replicative aging is caused by progressive accumulation of extrachromosomal rDNA

circles (ERC s) in yeast mother cells. According to this model, ERC s are produced

stochastically by intrachromosomal homologous recombination at the rDNA locus and

are inherited asymmetrically by mother cells, which leads to ERC accumulation and

replicative senescence. The rDNA locus in S. cerevisiae consists of a tandem array of

~150, 9.1 kb direct repeats, each of which encodes the four rRNAs (18S, 5.8S, 25S, and

5S) in precursor form. Many aspects of the ERC model have been supported

experimentally. Numerous studies support the view that ERC s are produced by

homologous recombination, are self-replicating, are inherited asymmetrically, and

accumulate in mother cells (45,54,55).

More controversial is the role ERC s play in the aging process. Are ERC s

"mediators" or "markers" of yeast aging? Certain findings link ERC production with

regulation of life span and support a "mediator" role for ERC s. One of the first life span

extending mutations characterized in yeast (SIR4-42) was found to redirect "silent

information regulator" (Sir) protein complexes to the rDNA locus and limit

recombination (5 1,56). Expression of SIR2, which encodes a nucleolar NAD-dependent

histone deacetylase, correlates with longevity. Sirdp binds to rDNA and suppresses

rDNA recombination and ERC production (57-59). Deletion of SIR2 shortens life span,

whereas overexpression of SIR2 extends life span (60). FOB1 encodes a nucleolar "fork

blocking" protein that binds to the replication fork barrier (RFB) site in rDNA and in so

doing halts DNA replication in the direction opposite of pre-3 5S rRNA transcription (61-

63). The RFB site and the overlapping HOT1 site promote rDNA recombination (63,64).

Mutations in (or deletion of) FOB1 reduce rDNA recombination, lower ERC levels, and









extend life span (65). Recombination of replication forks stalled at RFB sites is

suppressed by Sirdp (66), which partly explains the role of Sir2-dependent silencing in

extending life span. Also, introduction of a plasmid carrying a stretch of rDNA, as an

"artificial" ERC, was shown to reduce life span (54).

On the other hand, ERC s have been interpreted as a "marker" of aging that are a

consequence, not a cause, of aging. Mutations that impair DNA replication,

recombination, or repair have been observed to reduce life span without concomitant

accumulation of ERC s (67-69). However, reduction in life span may be the result of the

combined effects of age-dependent and age-independent processes at work in certain

mutants. The hrmlA mutants, which affect rDNA recombination, age prematurely due to

a combination of the normal aging process and a G2-like cell cycle arrest (69). Similarly,

sgs1 mutants exhibit a shortened life span because of the combined effects of the normal

aging process and cell cycle arrest due to defective recombination (70). Some petite

mutants have been shown to have elevated ERC levels (71), but extended life spans (72).

However, to our knowledge, both elevated ERC levels and extended life span in petite

mutants have not been demonstrated side-by-side in the same strain. A sir2 mutant with

an extended life span was reported to have normal ERC levels (73). More generally, the

effects of SIR2 on life span have been attributed to altered patterns of gene expression,

including altered transcription of rDNA, which may lead to an imbalance in ribosome

synthesis (74,75). Thus, although there is agreement that the rDNA locus plays a key

role in the yeast aging process, the precise role of extrachromosomal DNAs remains

controversial.









To shed light on this controversy, we have developed a plasmid-based model

system to investigate the role of episomal DNAs in reduction of yeast life span. Here we

present the first comprehensive test of the ERC model of yeast aging proposed by

Sinclair and Guarente (54). We constructed three types of recombinant plasmid for this

purpose: ARS plasmids, ARS/CEN plasmids, and 2 CI origin plasmids. ARS plasmids

are most like ERC s in that they are circular DNA molecules with a replication origin but

lack a cis-acting partitioning sequence. Classic pedigree analysis studies by Murray and

Szostak showed that ARS plasmids exhibit a strong bias to be retained in mother cells

during mitosis (43). Thus, ARS plasmids are predicted to accumulate in mother cells like

ERC s, but this has not yet been demonstrated. ARS/CEN plasmids contain a

centromeric DNA region that acts in cis to attach plasmid DNA to the mitotic spindle and

ensure efficient delivery to daughter cells during mitosis. ARS/CEN plasmids should not

accumulate in mother cells. 2 CI origin plasmids typically contain a DNA replication

origin, a cis-acting REP3 STB element, and one copy of an inverted repeat that regulates

plasmid copy number (~20 to 40 copies/cell) (76). The REP3 STB element actively

partitions plasmid DNA to daughter cells during mitosis in cil- yeast strains (i.e., in

strains that contain the endogenous 2 CI circle DNA plasmid that encodes proteins that

interact in transrt~t~rt~t~rt~t~rt~ with REP3 STB) (76). 2 CI origin plasmids are not predicted to

accumulate in mother cells, although the 2 CI plasmid partitioning machinery is not

predicted to exhibit the fidelity of a centromere-based partitioning machinery. We also

constructed a series of plasmids containing functional rDNA repeat units, and tested their

effects on life span. This represents a significant improvement over a previously reported









experiment (54), which employed a non-functional stretch of rDNA (i.e., rDNA

incapable of being transcribed to yield full-length 35S pre-rRNA).

Roles of Different Cis-Acting Plasmid Sequences in Reduction of Yeast Replicative
Life Span

To study the effects of plasmids on yeast replicative life span, we generated two

series of plasmids based on commonly-used integrating vectors-pRS306 and pRS305

(77). In each plasmid, we inserted ARS1, or ARS1~ and CEN4, or the 2 CI circle origin (see

Materials and Methods). ARS1~ (autonomous replicating sequence 1) is a nuclear

genomic DNA replication origin whose function and domain organization have been

studied in detail (78). Centromeric DNA from chromosome IV (CEN4) has been mapped

and functionally dissected (79). The region of the 2 CI circle plasmid extending from

Table 2-1. Plasmids used in this study.
Plasmid Origin, Insert Marker Backbone

pJPA105 2 CI, rDNA repeat (Xmal endpoints) 7RP1 pAFl15
pJPA106 2 CI, rDNA repeat (Ahdl endpoints) 7RP1 pAFl15
pJPA107 2 CI, rDNA repeat (Psil endpoints) 7RP1 pAFl15
pJPAll3 ARS1~ GRA3 pRS306 (77)
pJPAll4 rDNA ARS GRA3 pRS306
pJPAll6 ARS1~, CEN4 GRA3 pRS306
pJPAll7 rDNA ARS, CEN4 GRA3 pRS306
pJPAl38 2 CI GRA3 pRS306
pJPAl33 ARS1~ LEU2 pRS305 (77)
pJPAl36 ARS1~, CEN4 LEU2 pRS305
pJPAl48 2 CI LEU2 pRS305
REP3 through the adj acent 599 bp 2 CI repeat functions as a replication origin as well as a

cis-acting plasmid partitioning element (76,80). The plasmids used in this study are

summarized in Table 2-1.









To evaluate effects on life span, plasmids were transformed into strain W303AR5

(54). For each plasmid, six independently-isolated transformants were analyzed in

parallel, and each life span curve reflects their collective behavior. Selection for the

plasmid was maintained during life span analysis. Virgin mother cells unable to give rise

to 5 daughters were discarded to exclude contributions from mother cells without

plasmid. To identify mother cells that stopped dividing due to plasmid loss, rather than

senescence, cells that had not divided in 2 days were transferred to nonselective medium

and monitored for cell division and colony formation. A low percentage (<10%) of

mother cells were found to give rise to colonies, and were excluded from the life span

data set. Life span plates were incubated during the daytime at 300C, but placed

overnight (~12 hours) at 140C, which gave a slightly, but significantly (p<0.01) longer

life span than observed on plates stored overnight at 40C (Figure 2-1A).

Interestingly, transformants harboring pJPAll3 (ARS1) showed dramatic reductions in

both average and maximum life span compared to the Ura+ control strain yAF6 (Figure

2-1B). yAF6 differs from pJPAll13 transformants only in terms of plasmid DNA

topology (i.e., integrated in yAF6 and episomal in transformants). Transformants

containing pJPAll6 (ARS1~, CEN4) have a reduced average life span compared to yAF6,

but exhibit a maximum life span similar to yAF6 (Figure 2-1B). Thus, addition of a

CEN4 element to an ARS1~ plasmid suppresses reduction in maximum life span, but does

not completely compensate for, or protect against, effects on average life span. Plasmids

containing the 2 CI circle origin of replication were also constructed and analyzed. Yeast

cells harboring pJPAl38 (2 CI ori) show a reduction in both average and maximum life









span (Figure 2-1B). Generally speaking, the extent of reduction in average and

maximum life span in pJPAl38 (2 CI ori) transformants is intermediate between that


Figure 2-1.Life span analysis of plasmid-transformed yeast. Number of daughter cells
(generations) produced per mother cell are plotted as a function of mother cell
viability. A) Life span curves of strain W303AR5 (54) grown on SD
(synthetic dextrose) and S+D (dextrose added after autoclaving) media at
300C during the daytime and stored overnight (~12 hours) at 40C or 140C.
The number (n) of mother cells analyzed per curve is as follows: SD 40C,
n=60; SD 140C, n=59; S+D 140C, n=60. B) Life span curves of W303AR5
transformed with plasmids pJPAll3 (ARS1~), pJPAll6 (ARS1~, CEN4), or
pJPAl38 (2 CI ori), and control strain yAF6 (URA3) (n=55, 47, 57, and 58,
respectively). C) Life span curves of W303AR5 transformed with plasmids
pJPAl33 (ARS1), pJPAl36 (ARS1, CEN4), pJPAl48 (2 CI ori), and control
strain yAF5 (LEU2) (n=38, 33, 41, and 59, respectively). D) Life span curves
of W303AR5 transformed with pJPAll16 (ARS1~, CEN4) determined on SD
and YPD (n=45 and 49, respectively). Life spans of control strains yAF6 and
W303AR5 were determined on YPD (n=50 and 55, respectively). Plasmids
are described in Table 2-1.

observed in pJPAll3 (ARS1) and pJPAll6 (ARS1~, CEN4) transformants (Figure 2-1B).

The results from multiple life span experiments are summarized in Table 2-2.












Table 2-2. Life span data summary.
Plasmid/Strain Mean Life Span Maximum Life Span n*"

pJPAll3 12.4 & 1.8 21.8 & 2.2 4
pJPAll6 23 A 1.4 39 & 2.7 4
pJPAl38 16.3 A 1.8 31.3 & 0.6 3
yAF6 33.2 & 3.0 42 & 1 3
The results reported above were obtained with plasmids carrying a GRA3 selectable

marker. To eliminate the possibility that effects of plasmids on life span were due to

GRA3 or medium lacking uracil, we constructed plasmids with a LEU2 selectable marker

(Table 2-1), and conducted life span experiments on medium lacking leucine. The results

obtained with the LEU2 plasmid series were very similar to results obtained with the

GRA3 plasmid series (Figure 2-1C). pJPAl33 (ARS1~) caused dramatic reductions in

average (9.9 generations) and maximum (17 generations) life spans compared to the Leu+

control strain yAF5. yAF5 yielded an average (30.3 generations) and a maximum (44

generations) life span very similar to the average and maximum lifespan for yAF6 (Table

2-2). Transformants containing pJPAl36 (ARS1~, CEN4) yielded a maximum life span of

38 generations, but an average life span of 24 generations, similar to what was observed

for the GRA3 plasmid pJPAll6 (ARS1, CEN4). Plasmid pJPAl48 (2 CI ori) reduced the

average (15.5 generations) and maximum (31 generations) life span to an extent

intermediate between pJPAl33 (ARS1~) and pJPAl36 (ARS1, CEN4) (Figure 2-1C),

similar to what was observed with the GRA3 plasmid pJPAl38 (2 CI ori) (Figure 2-1B).

The reduction in average life span by ARS1, CEN4 plasmids pJPAll6 and

pJPAl36 was unexpected. A similar plasmid had previously been reported to have no

effect on life span when grown on YPD medium (54). One possible explanation for this









difference was that ARS1, CEN4 plasmids are occasionally lost from mother cells,

causing them to cease division on selective medium prior to senescence, which would

result in a reduction in average life span. To test this, ARS1, CEN4 plasmid

transformants were analyzed on non-selective YPD medium as done previously (54). On

YPD, transformants carrying pJPAll6 (ARS1~, CEN4) were as long-lived as control

strains yAF6 (URA3) and W303AR5 (Figure 2-1D). pJPAll6 transformants analyzed in

parallel on selective SD medium showed a reduction in average life span (Figure 2-1D),

as expected. These findings support the interpretation that ARS1~, CEN4 plasmids, which

are present at near-unit copy number in transformants (see below), are occasionally lost

from mother cells, rendering them unable to divide at a point in their life span prior to

normal senescence.

We have also examined the effects of two well-known plasmids that carry the

7RP1 selectable marker. pTV3 carries the 2 CI origin whereas pRS314 carries ARSH14

and CEN6 (77,80). Life spans of transformants containing each plasmid were analyzed

on medium lacking tryptophan. pTV3 transformants had an average life span of 18.7 and

a maximum life span of 32, both values of which are in good agreement with

corresponding values for the 2 CI origin plasmids pJPAl38 and pJPAl48 (Figure 2-1 and

Table 2-2). pRS3 14 had average and maximum life spans of 21 and 41, respectively,

which are in good agreement with values obtained with the ARS1~/CEN4 plasmids

pJPAll6 and pJPAl36 (Figure 2-1 and Table 2-2). These data allow us to exclude a

specific role for ARS1~ and CEN4 in life span reductions presented above (Figure 2-1).

Plasmid Inheritance Correlates with Reduction in Yeast Life Span

The plasmids used in this study were constructed to explore relationships between

plasmid inheritance and effects on life span. Mitotic stability and plasmid copy number









are widely-used measures of plasmid DNA inheritance. Mitotic stability is defined as the

proportion of a population of cells grown under selection that contains plasmid. We

determined the mitotic stability and plasmid copy number of the plasmids used in life

span experiments. Included in our studies were plasmids containing the rDNA ARS.

rDNA repeats contain a single, relatively weak ARS (81). pJPAll4 and pJPAll7

contain the rDNA ARS at the same position as ARS1 in pJPAll3 and pJPAll6,

respectively (see Table 2-1 and Materials and Methods).

Plasmid pJPAll3 (ARS1~) was found to have a mitotic stability of approximately

20% (Figure 2-2A), which is typical of yeast replicating plasmids containing ARS1,

which exhibit a mother cell partitioning bias (43). pJPAll6 (ARS1~, CEN4) exhibited a

much higher mitotic stability, ~90%, which is consistent with the presence of CEN4

centromeric DNA, and agrees with the mitotic stability of pRS316 (ARSH4, CEN6)

(Figure 2-2A). pJPAl38 (2 CI ori) showed a high degree of mitotic stability, ~90%

(Figure 2-2A). The 2 CI origin plasmid pRS424 had a somewhat lower mitotic stability

by comparison (Figure 2-2A). pJPAll4 (rDNA ARS) has a very low mitotic stability,

<1% (Figure 2-2A). The presence of CEN4 with the rDNA ARS in pJPAll17 improves

mitotic stability to ~3 5% (Figure 2-2A). These results with pJPAl l4 and pJPAl l7 are

consistent with the low efficiency of the rDNA ARS (81). Not surprisingly, it was

impractical for us to carry out life span analyses of transformants containing pJPAll14.
























Figure 2-2.Plasmid inheritance studies. Plasmids are denoted by cis-acting elementss.
See Table 1 for plasmid descriptions. pRS316 (ARSH4, CEN6, (77)) and
pRS424 (2 CI ori, 7RPl, (82)) are included for comparison purposes. A)
Mitotic stability determinations. Mitotic stability is defined as the percentage
of colony forming units in a culture grown under selective conditions that
contains plasmid-borne selectable marker. Side-by-side bars are
determinations from separate experiments. Average and standard deviation
values are plotted. B) Plasmid copy number in toto for cell population.
Average and standard deviation values from Southern blots of genomic DNA
digested with BamHI (filled bars) and PstI (open bars) are shown. C) Plasmid
copy number on a per cell basis. Values were calculated by dividing copy
number values from panel B by mitotic stability values from panel A (average
of both experiments). The variances in copy number values were determined,
assuming a log normal distribution of values. Variances for all values were
near 1.0, with the exception of rDNA ARS plasmid copy number, which had a
variance of 3.0, which is indicative of a higher level of error in this
measurement.

Plasmid copy number was determined using Southern blot analysis. Copy number

was displayed either as the total number of plasmids compared to the total number of

genomes (copy number in the population, Figure 2-2B) or the total number of plasmid

compared to the fraction cells (genomes) that contain a copy of the plasmid (copy number

per cell, Figure 2-2C) by using a plasmids mitotic stability. Copy number determinations

using two different restriction enzymes gave comparable results (Figure 2-2B). pJPAll3

(ARS1) exhibited the highest plasmid copy number (Figure 2-2C). Plasmids pJPAll6

(ARS1, CEN4) and pJPAll7 (rDNA ARS, CEN4) exhibited near-unit copy number









values (Figure 2-2C), which is typical of centromeric plasmids (79), such as pRS316

(ARSH4, CEN6) (77). pJPAl38 (2 CI ori) exhibited a copy number of ~33 (Figure 2-2C),

which is in the range of copy number values reported for other 2 CI origin plasmid vectors

(80). The high copy number of pJPAll13 is primarily due to the asymmetric inheritance

of this plasmid and its accumulation in mother cells, rather than ARS strength per se. We

reach this conclusion because pJPAll4, which contains a weak (rDNA ARS) replication

origin, achieves a copy number almost as great as pJPAll3, which contains a strong

(ARS1) replication origin (Figure 2-2C). Thus, pJPAll3 demonstrates a correlation

between extent of reduction of transformant life span (Figure 2-1B) and tendency to be

inherited asymmetrically and attain a high copy in yeast cells (Figure 2-2C).

Plasmids Do Not Significantly Increase ERC Levels

The results presented above suggest that reduction in life span by the ARS1~ plasmid

pJPAll3 is due to asymmetric inheritance and accumulation in mother cells. An

alternative explanation is that pJPAll3 increases ERC levels in transformed cells, and

thereby reduces life span indirectly. To address this possibility, we measured

recombination at the rDNA locus using an ADE2 marker loss assay and measured ERC

levels in transformed cells by Southern blotting.

To analyze the frequency of recombination at the rDNA locus, we took advantage

of the fact that W3 03AR5 contains ADE2 integrated at the rDNA locus (54).

Recombination between flanking rDNA repeats results in loss ofADE2 and a change in

colony color. The frequency of half-red sectored colonies is a measure of rDNA

recombination rate (events per cell division). Transformation of yeast with plasmid

results in a small increase in rDNA recombination as measured by ADE2 marker loss.

For W303AR5, we find that ADE2 marker loss occurs at a frequency of ~1.3 per









thousand cell doublings (Figure 2--3A), which is in good agreement with frequencies

reported by others (60,68,69). The rate ofADE2 marker loss from yAF6 (URA3) occurs

at ~2.7 per thousand (Figure 2-3A). Transformants containing the three plasmids used in

this study, pJPAll3 (ARS1), pJPAll6 (ARS1, CEN4), and pJPAl38 (2 CI ori), exhibited

marker loss rates of 4.1i, 4.5, and 4. 1 per thousand cell doublings, respectively. The

differences between transformants and yAF6 represent increases of less than 2-fold.

Higher levels ofADE2 marker loss are typically observed in strains with reduced life

spans. For example, short-lived sir2A mutants exhibit ADE2 marker loss rates >10-fold

higher than isogenic SIR2 strains (60).

To directly compare ERC levels, yeast transformants and control strains were

analyzed by Southern blotting, and ERC monomer bands were quantitated (see Materials

and Methods). ERC monomers consist of a single 9. 1 kb rDNA repeat and were chosen

for purposes of quantitation because they are well-resolved from chromosomal rDNA and

other ERC bands on Southern blots. ERC monomer levels in transformants were not

significantly different than ERC monomer levels in control strains. Control strains

W303AR5 and yAF6 (URA3) have approximately 0.0007 and 0.0015 ERC monomers

per total chromosomal rDNA, respectively (Figure 2-3B). Transformants bearing

pJPAll3 (ARS1), pJPAll6 (ARS1, CEN4), and pJPAl38 (2 CI ori) have ERC monomers

levels of 0.0014, 0.001, and 0.001, respectively (Figure 2-3B). These values are within

the error of measurements and are not significantly different (Figure 2-3). For

comparison, we examined yAF5 (LEU2), which contains a copy of pRS305 integrated at

the leu2-113 locus, and found that the ERC monomer level was 0.001, which is

intermediate between W303AR5 and yAF6 (Figure 2-3B). Quantitation of slower-









migrating ERC multimer bands did not reveal significant differences in levels between

transformant and control strains (data not shown). We conclude that plasmids do not

have a significant effect on ERC levels.














Figure 2-3. Extrachromosomal rDNA circle (ERC) formation in yeast transformants.
Plasmids are denoted by cis-acting elementss. See Table 2-1 for plasmid
descriptions. Control strains W303AR5 (W303), yAF5 (LEU2), and yAF6
(URA3) did not contain plasmid. A) ADE2 marker loss assay. The number of
half-red sectored colonies on minimal selective medium per total colony
number defines the per (first) cell division rate of loss of the ADE2 marker
from the rDNA repeat in W303AR5. Total number (n) of colonies scored is
shown. B) ERC monomer levels. Southern blotting analyses of DNA from
transformed and control strains grown on selective media were done to
quantify chromosomal rDNA and ERC monomer band levels (see Materials
and Methods). ERC monomer band intensity was divided by chromosomal
rDNA band intensity to give a normalized ERC monomer/chromosomal
rDNA ratio. Average and standard deviation values are plotted.

Plasmid Accumulation Correlates with Reduction in Life Span

If plasmids reduce life span in a manner analogous to ERC s, then plasmid DNAs

should accumulate in old mother cells. To test this prediction, we used a biotinylation

and magnetic sorting approach to isolate ~?-generation old yeast cells (see Materials and

Methods). Plasmid DNA levels in young and old cells were measured by quantitative

Southern blotting.

The ages of old and young (unsorted) cells were determined by counting bud

scars stained with Calcofluor (83). From single sort experiments, the average ages of









yeast transformed with pJPAll3, pJPAll6, pJPAl38, and yAF6 were 6.9, 7.0, 6. 1, and

6.2 generations, respectively (Figure 2-4A). Young cells from the same cultures were an

average of 1.5, 1.4, 1.1, and 1.1 generations old, respectively (Figure 2-4A). Inspection

of the Southern blot clearly reveals increases in relative amounts of pJPAll13 (ARS1) and

pJPAl38 (2 CI ori) in old cells (Figure 2-4B). pJPAll6 (ARS1~, CEN4) did not

accumulate in old cells, and yields bands similar in their intensities to corresponding

bands from yAF6 (Figure 2-4B). In a striking illustration of the accumulation of

pJPAll3 and pJPAl38 in old cells, the linearized plasmid DNA bands can be observed

by ethidium bromide staining (Figure 2-4D). ERC levels in young and old cells were

also analyzed by Southern blotting. Hybridization to rDNA probe revealed ERC bands

and a broad band corresponding to the rDNA locus on chromosome XII (Figure 2-4C).

We note that all old cell preparations contained increased numbers of both monomeric

and slower-migrating ERC species (Figure 2-4C). The ERC and rDNA repeat bands

collapse to a single 9.1 kb band following digestion with KpnI, which cuts rDNA once

(data not shown).

Chromosomal and plasmid band intensities were quantitated using a

PhosphorImager. Consistent with our determinations in Figure 2-2, pJPAll3 (ARS1) and

pJPAl38 (2 CI ori) are present at high copy number in young cells, but pJPAll6 (ARS1,

CEN4) is not (Figure 2-4E). In ~?-generation old transformants, the plasmid copy

numbers for pJPAll3 and pJPAl3 8 are dramatically increased, reaching values of 254

and 137, respectively (Figure 2-4F). This represents a difference in copy number

between young and old cells of ~13-fold for pJPAll3 and~-6-fold for pJPAl38. By





Figure 2-4.Plasmid DNA and extrachromosomal rDNA circle (ERC) levels in young and
old cells. Panel A conveys the cis-acting elements present in each plasmid
(see also Table 2-1). Plasmids are abbreviated by numbers in panels B-H. All
plasmids carry UR3. Control strain yAF6 (UR3) did not contain plasmid.
Old cells were harvested using a biotinylation and magnetic sorting approach
(see Materials and Methods). A) Age profile histograms of young and old
cells. Number of cells is plotted as a function of number of bud scars (n>40
for each histogram). B) Southern blot of plasmid DNAs. Pstl-digested
genomic DNA yields a 3.67 kb UR3 band. Other bands are plasmid-derived.
Genomic UR3 DNA in lane "Old 113" migrated as two bands due to partial
over-digestion of this sample. C) Southern blot of ERCs. D) Ethidium









bromide stained agarose gel corresponding to the blot in panels B and C.
DNA marker sizes (in kb) are shown. E and F. Plasmid levels in young and
old cells (quantitation of data presented in panel B). G and H) ERC
monomer levels in young and old cells (quantitation of data presented in panel
C). For E-H, ratios of episome plasmidd or ERC monomer) band intensity
divided by chromosomal rDNA band intensity (X1000) are plotted (on a semi
log scale). See Fig. 2-1B for corresponding life span data. Comparable
results were obtained from similar cell sorting and Southern blotting
experiments and are discussed in the Results section.

comparison, 7-generation old pJPAll6 transformants show no significant increase in

plasmid copy number (Figure 2-4F).

In a separate experiment with pJPAll3 and pJPAll6 transformants, in which

genomic DNA was digested with BamHI instead of Pstl, quantitative analysis revealed

that young cells contained 27 and 1.5 plasmids/cell, respectively, whereas old cells

contained 283 and 1.2 plasmids/cell, respectively (data not shown). This corresponds to a

~10-fold increase in plasmid copy number for pJPAll3 in~?7-generation old cells, and no

significant increase in pJPAll6 copy number, which agrees with findings presented in

Figure 2-4E, F.

ERC monomer levels were also quantitated in young and ~?-generation old

transformants and yAF6. ERC monomer levels in young cells were equal or close to

0.001 (Figure 2-4G), which agrees with measurements presented above (Figure 2-3B). In

old cells, however, ERC monomer levels were appreciably higher, and exhibited

increases between ~20-fold to~-70-fold (Figure 2-4H). The levels of ERC s we observe

in ~?-generation old cells appears comparable to ERC levels in sorted cells of similar

age reported by others (e.g., (54,60)), although quantitative analysis of ERC levels in

young and old yeast cells is not commonly reported in the literature.

In the experiment shown in Figure 2-4, ERC monomer levels in yAF6 (URA3) are

higher than in transformants (Figure 2-4H). This raises the question: does the presence










of plasmid reduce ERC levels? In a separate experiment, ERC monomer levels in

young cells were equal or close to 0.001 (ERC monomer/chromosomal rDNA) and ERC

monomer levels in old transformants containing pJPAll3, pJPAll6, and pJPAl38 and in

old yAF6 cells were determined to be 0.083, 0.075, 0.045, and 0.081, respectively (data

not shown). The similar ERC levels in yAF6 and transformants in this experiment

suggest that plasmid vectors do not appreciably affect ERC monomer levels (Figure 2-5).

Does the extent of ERC accumulation in old cells in Fig. 4 agree with predictions

based on our estimates of rates of recombination within the rDNA locus (see above,

Figure 2-3)? If we assume that extrachromosomal rDNA repeats are generated at a rate

of 0.5 per cell per generation, and that ERC s are retained in mother cells, then 6-7

generations should yield an increase of between 32- to 64-fold, which is similar to the

observed range of increase from 20- to 70-fold (Figure 2-4G, H).

We have also quantitated the relative amount of all ERC s (i.e., monomers,

multimers, and concatemers) found in old transformants containing pJPAll3, pJPAll6,

and pJPAl38 and in old yAF6 cells. We found levels, respectively, of 0. 140, 0. 136,

0.086, and 0.238 (extrachromosomal rDNA/chromosomal rDNA; data not shown). These

values mirror levels of accumulation of ERC monomers presented in Figure 2-4H. Thus,

ERC monomers comprise approximately 1/4 to 1/3 of all extrachromosomal rDNA

repeats and are present at similar levels relative to all ERC s in old transformed and

untransformed cells.

To extend these studies, yeast sorting experiments were done with transformants

containing the LEU2 plasmids pJPAl33 (ARS1~), pJPl36 (ARS1, CEN4), and pJPAl48 (2

CI ori), and with the LEU2 strain yAF5. The average ages of sorted yeast transformed









with pJPAl33, pJPAl36, pJPAl48, and yAF5 are 7.1, 7.0, 7.6, and 7.9 generations,

respectively (Figure 2-5A). Young cells from the same cultures were an average of 1.7,

1.0, 1.6, and 1.6 generations, respectively (Figure 2-5A). pJPAl33 and pJPAl48 attain

copy number levels of 119 and 39, respectively, in ~?-generation old cells (Figure 2-5C).

This represents an increase in copy number between young and old cells of ~30- and ~8-

fold for pJPAl33 and pJPAl48, respectively. pJPAl36 did not show a significant

increase in old cells (Figure 2-5C). In comparison to pJPAll3 and pJPAl38, pJPAl33

and pJPAl48 reached lower absolute levels of plasmid in ~?-generation old cells.

However, pJPAl33 and pJPAl48 accumulated to similar extents in terms of fold-

increase. To resolve if this difference in absolute levels of plasmids in old cells was due

to experimental error, sorting experiments with transformants and the control strain were

repeated, followed by Southern analyses. The repeat experiment gave results very similar

to first experiment, both in terms of absolute level of plasmid in young and old cells as

well as fold-increase in young and old cells (Figure 2-5B, C). This indicates that

plasmids with identical ARS1~ origins and CEN4 elements, but with different backbones

and selectable markers, are maintained at different absolute copy number levels in young

and old cells. Nevertheless, similar fold-differences in plasmid levels are observed

between young and ~?-generation old cells. This indicates that ASR1 and CEN4 elements

present on plasmids functionally determine patterns of plasmid inheritance and

accumulation during yeast mother cell replication.

Next, ERC monomer levels in transformants containing pJPAl33, pJPAl36, and

pJPAl48, and in strain yAF5 were quantitated. In young cells, ERC monomers were

detected at relatively high levels (Figure 2-5C). However, ERC monomer levels in ~7










generation old cells were similar to levels observed above for pJPAll3, pJPAll6, and

pJPAl38 transformants (compare Figures 2-4H and 2-5D). Thus, ERCs in the Leu+

transformants showed accumulation over a range of ~3-fold to ~12 fold between young

and old transformants. This range of fold-increase is approximately 7-fold lower than the

~20-fold to ~70-fold increase in ERC levels between young and old Ura+ transformants.

This suggests that the rate of ERC accumulation during the aging process is regulated so

that old cells of similar ages contain similar levels of ERC s despite differences in initial

levels of ERC s in young cells.























Figure 2-5.Plasmid DNA and extrachromosomal rDNA circle (ERC) levels in young and
old cells. Plasmids are abbreviated by numbers in panels B-E. Panel A
conveys the cis-acting elements present in each plasmid (see also Table 2-1).
All plasmids carry LEU2. Control strain yAF5 (LEU2) did not contain
plasmid. Data were collected as described in Fig. 4. A. Age profile
histograms of young and old cells. B and C. Plasmid levels in young and old
cells (semi log plot). Data from two Southern blotting experiments are shown
(Exp 1 and Exp 2). D and E. ERC monomer levels in young and old cells
(semi log plot). See Fig. IC for corresponding life span data.









An important trend emerges from our studies of plasmid accumulation in old cells.

Plasmids that accumulate to the greatest degree in old cells (Figures 2-4 and 2-5) exert

the most profound effect on life span (Figure 2-1). ARS1 plasmids attain the highest copy

numbers in old cells and have the most pronounced effect on life span. ARS1 /CEN4

plasmids maintain a copy number near unity in young and old cells and have a small

effect on maximum lifespan and a moderate effect on average life span. Plasmids with 2

CI origins attain a copy number in old cells roughly half that of ARS1 plasmids and reduce

life span roughly half as much as ARS1 plasmids. This suggests the existence of an

inverse relationship between plasmid accumulation in old cells and reduction in yeast life

span.

Terminal Cell Morphology

Currently, in the field of yeast aging, there are few approaches available to directly

address the senescent phenotype in old non-dividing cells. To address this issue

indirectly, we scrutinized the "terminal" morphology of cells at the end of their life span

(Appendix A-H). The rationale for this approach is that cell morphology is a phenotypic

indicator of cell cycle stage and can serve as a basis to compare senescent cells (70). If

cell morphology in terminal transformed cells is very different from the morphology of

terminal wild type cells, this would imply that different mechanisms may bring about the

senescent phenotype in transformed and untransformed cells.

To examine terminal yeast cells, images of terminal cells were collected from

three different life span experiments. Three different cell morphologies were scored:

unbudded cells, single-budded cells with small buds, and single-budding cells with large

buds (70). Bud emergence in S. cerevisiae correlates with entrance into S phase, and









small buds are indicative of early S phase, whereas large buds are indicative of late S/G2

or mitotic arrest. Unbudded cells are in G1 phase. Between 10-15% of the terminal cells,

transformed or untransformed, had multiple buds (data not shown) and were omitted

from this comparison. For pJPAll3 (ARS1) and pJPAll6 (ARS1~, CEN4) transformants,

and W303AR5, more than 50% of terminal cells were unbudded (Figure 2-6). Typically,


Figure 2-6. Terminal morphology of senescent cells. Cells at the end of life span
experiments were classified according to budding pattern as described (70).
Small buds were defined as having a diameter less than 25% of the diameter
of the mother cell. All other buds were classified as large. Average and
standard deviation values from three independent experiments are shown (n
>40 for each transformant or control strain in each experiment).

between 50% and 60% of senescent yeast cells have been found to be unbudded (69,70).

pJPAll6 transformant cells consistently yielded the highest proportion (~65%) of

unbudded cells (Fig. 6). yAF6 (URA3) and pJPAl38 (2 CI ori) transformants ceased

dividing with a predominance, yet a lower percentage, of unbudded cells (Figure 2-6).









Thus, the maj ority of pJPAll13 transformants, like W303AR5 cells, senesced in G1, as

expected. In addition, similar proportions of small budded and large budded terminal

cells in senescent pJPAll3 transformants and W303AR5 cells (Figure 2-6) indicate that

similar proportions of these cells arrested in similar phases (S or G2 M) of the cell cycle.

Thus, this analysis supports the interpretation that pJPAll3 (ARS1) reduces life span by a

normal aging process.

Do Functional rDNA Transcriptional Units Play a Role in Reduction in Life Span?

Although plasmids without rDNA sequences reduce yeast life span, it is important

to consider a potential role for rDNA sequences in life span reduction. It is possible that

ERC s reduce life span in a manner that is mechanistically more complex than the

manner in which plasmid episomes reduce life span. There are significant differences in

coding potential between plasmids and ERC s. The 9. 1 kb rDNA repeat carries genes for

rRNA precursors as well as the gene TAR1, which lies on the strand opposite the 25S

rRNA and encodes a mitochondrial protein (84). One way to address this issue is to ask

whether or not a plasmid vector carrying an rDNA repeat unit has a more pronounced

effect on life span than plasmid vector alone. It is important to note this issue was not

completely addressed in a previous study employing the rDNA-containing plasmid

pDS163 (54). Plasmid pDS163 does not contain a functional 9.1 kb rDNA repeat unit.

The rDNA on pDS163 consists of a 12. 1 kb insert extending from an EcoRI site within

the coding sequence of 5.8S rRNA to the 5'-most EcoRI site in the 25S rRNA coding

region (data not shown). The 12.1 kb fragment does not carry a full-length 35S pre-

rRNA transcription unit and is capable of producing only a truncated 35S pre-rRNA

transcript, which if processed would be incapable of yielding mature 25 S rRNA.









To determine if an episomal rDNA repeat influences life span, we constructed

three plasmids containing 9.1 kb rDNA repeats and used them in life span experiments.

The three plasmids, pJPA105, pJPA106, and pJPA107 contain 9.1 kb repeats with

different endpoints in the plasmid pAFl5, which contains a 2 CI origin (see Materials and

Methods, and Table 2-1). Plasmid pJPA105 contains a repeat with Xmal end points,























Figure 2-7.Life span analysis of yeast transformed with plasmids containing rDNA
repeats. Number of daughter cells (generations) produced per mother cell are
plotted as a function of mother cell viability. Life span analysis was done as
described in Figure 2-1 using W303AR5 carrying plasmids pJPA105 (n=45),
pJPA106 (n=43), or pJPA107 (n=46) and control plasmid pAFl15 (n=46).
Plasmids pJPA105, pJPA106, and pJPA107 contain full length (9.1 kb),
rDNA repeats with different endpoints (see Table 2-1 and Materials and
Methods). pJPA105 contains an rDNA insert with Xmal endpoints, which has
been shown to be functional in vivo (85).

which has been shown by Nomura and colleagues to functionally complement an rDNA

deletion in vivo (85). pJPA106 and pJPA107 contain repeats with Ahdl and Psil

endpoints, respectively, which should not interfere with rDNA gene expression (Figure 1-

2). A 2 CI origin plasmid was used because plasmids constructed with rDNA inserts









whose replication relied solely on the rDNA ARS were found to integrate into the

chromosomal rDNA locus with high frequency (as determined by Southern blot analysis;

data not shown). Life span determinations of W303AR5 transformants containing

pAFl15, pJPA105, pJPA106, and pJPA107 were done as described above (see Figure 2-

1). pJPA105, pJPA106, pJPA107, and pAFl5 transformants gave very similar life span

curves, indicating that the presence of a functional rDNA repeat does not have a dramatic

effect on life span (Figure 2-7). All four plasmids affect life span to an extent similar to

the 2 CI origin plasmids pJPAl38 and pJPAl48 (Figure 2-1B, C), although the average

life spans for pJPA105, pJPA106, pJPA107, and pAFl15 (13.3, 11.8, 11.7, and 12.2

generations, respectively) are lower than the average life spans for pJPAl38 and

pJPAl48 transformants (15.5 and 16.3 generations, respectively; Figure 2-1 and Table 2-

2). Life span curves for pJPA106 and pJPA107 transformants did not show a statistically

significant difference from pAFl5 transformants based on the Wilcoxon signed pair rank

test (p>0.05) Only transformants carrying pJPA105 and pAFl5 exhibited a statistically

significant difference (p<0.05), but this represents a small increase in life span of

transformants carrying pJPA105. These findings support the conclusion that the presence

of a full-length rDNA repeat per se does is not required for, and does not necessarily

augment, reduction in yeast life span.

Summary

Our studies show that yeast plasmids accumulate in mother cells and reduce

replicative life span. The effect of plasmids on life span appears to be a direct effect, and

not an indirect effect on ERC levels in mother cells. A functional rDNA repeat unit is

not required for reduction in life span, and the presence of a functional rDNA repeat does

not augment reduction in life span by plasmids. Thus, plasmids containing ARS










elements appear to "mimic" ERC-mediated reduction in life span. These findings

provide strong evidence that replicative aging in S. cerevisiae is caused by accumulation

of episomal DNA. The fact that functional rDNA sequences are not required for

reduction in life span argues that expression of rDNA genes present on ERC s is not a

causative process in yeast aging. This indicates that accumulation of episomal DNAs,

such as ARS plasmids and ERC s, is one mechanism by which yeast life span is

regulated.















CHAPTER 3
TWO MICRON CIRCLE: A NATURALLY OCCURRING EPISOME' S ROLE IN
AGING

One of the processes that has been proposed to regulate replicative life span in the

budding yeast Saccharomyces cerevisiae is the accumulation of extrachromosomal rDNA

circles (ERCs) by yeast mother cells (54). ERCs are generated by recombination within

the rDNA repeat region on chromosome XII and are passed on to daughter cells

infrequently due to an inheritance bias exhibited by replication origin-containing DNA

episomes (43). We have shown that plasmids containing an autonomously replicating

sequence (ARS; yeast DNA replication origin) reduce life span due to their accumulation

during replicative aging (86). This suggests that DNA episomes in general regulate

replicative aging, and reduce life span due to their accumulation in yeast mother cells.

The majority of laboratory strains of S. cerevisiae contain an endogenous plasmid

known as the two micron (2CI) circle, due to the length of its circular DNA determined by

electron microscopy (76,87). Strains harboring this non-Mendelian genetic element are

denoted cir ; strains lacking it are referred to as ciro (88). Four genes and multiple cis-

acting sequences on the 2 micron plasmid have been mapped and functionally dissected

(76,87). These are responsible for maintaining copy number at approximately 20-40

copies per cell by a recombination-based mechanism and ensuring high fidelity

transmission of the 2 micron plasmid during cell division and mating (76,87,89). There

are no significant growth phenotypes generally associated with the presence of the 2

micron plasmid in cir+ strains, and conversely, negligible growth advantages conferred to









ciro strains (76,87). This has led to the view that the 2 micron plasmid is a "parasitic"

DNA that imposes only a minor selective disadvantage to host strains (76,87). However,

previous studies have not examined the possibility of an effect of the 2 micron plasmid

on replicative life span. A minor effect on replicative life span is not predicted to result

in a discernable difference in vegetative growth rate, and may have been overlooked in

the past. To address this issue we have taken advantage of a novel and simple method for

curing a cir+ yeast strain of 2 micron plasmids. Previously described methods (90,91) for

curing strains of 2 micron plasmids are more time-consuming and are less convenient

than the method described herein.

New Method for Removal of Two Micron Circle

During the course of plasmid copy number studies published elsewhere (86), we

fortuitously observed that transformants containing recombinant yeast shuttle vectors

with an rDNA ARS lost 2 micron plasmid DNA more frequently than was expected,

based on the known inheritance behavior of 2 micron plasmids. In our initial Southern

blotting studies, half of the transformants containing plasmid pJPAll14 (4/8

transformants) or plasmid pJPAll8 (2/4 transformants) lost 2 micron plasmid DNA (data

not shown). Plasmids pJPAll4 and pJPAll8 are derived from pRS306 (77) and have

been described previously (86). pJPAll4 contains a 200 bp insert with the rDNA ARS;

pJPAll8 contains a complete 9. 1 kb rDNA repeat with its ARS. The rDNA ARS has

relatively weak replication origin activity due to the presence of a non-consensus ARS

consensus sequence (ACS) (81). As a result, the maj ority of pJPAll14 and pJPAll18

transformants form colonies slowly on selective SD medium. In instances where fast-

growing pJPAll8 colonies arose on transformation plates or streaks of individual

transformants, Southern analysis revealed that pJPAll8 had integrated into the rDNA









A B
M 1 2 3 4 5 6 7 8 9g d101112 1 2 3 4L 5 6 7 8 1011 12


Figure 3-1.Southern blot analysis of pJPAll14 transformants. Panel A. DNAs from
twelve transformants were digested with Pstl, separated on a 1% agarose gel
and stained with ethidium bromide. Size markers (M) are shown (in kb).
Panel B. DNAs from the gel in panel A were transferred to a nylon
membrane, hybridized to 32P-labeled 2 micron plasmid DNA probe, and
visualized with a PhosphorImager. The 6318 bp 2 micron plasmid contains
one PstI site and yields a single 6.3 kb band (arrowhead). The faint bands
above and below the 2 micron plasmid band likely correspond to
hybridization to regions of homology in yeast chromosomes (e.g., a region of
homology in Ch III yields a PstI fragment of 7747 bp, which corresponds to
the size of the upper faint band). Longer exposures of the Southern blot
revealed no detectable bands in lanes 8 and 9 (data not shown).

locus (data not shown). Transformants containing pJPAll8 were not studied further

because of the relative frequency with which pJPAll18 integrated into the rDNA repeat

locus. Fast-growing pJPAll4 transformants arose only very infrequently and were not

analyzed for plasmid integration by Southern blotting. Transformants containing plasmid

pJPAll3 (86), which is derived from pRS306 but contains the ARS1~ origin instead of the

rDNA ARS, did not show loss of 2 micron plasmids in our Southern blotting studies (data

not shown). ARS1~ contains an ARS consensus sequence (ACS) that conforms to the


lie~iC1)3dC~~









consensus observed in most ARS elements and is considered a strong ARS, unlike the

rDNA ARS.

These preliminary results suggested that pJPAll4 may be generally useful to cure

cir+ strains of 2 micron plasmid DNA. To further test the use of pJPAll14 for this

purp ose, we tran sform ed W30O3 AR5 with pJPAll 4, streaked i ndep endently -i sol ated

transformants to single colonies, obtained isolates lacking pJPAll4, and analyzed twelve

arbitrarily-chosen isolates by Southern blot analysis (see Materials and Methods).

pJPAll4 has two technical merits in this experiment. Because pJPAll4 contains the

rDNA ARS, it is readily lost from transformants grown in the presence of uracil. Loss of

pJPAll4 can be confirmed by growth on medium containing 5-fluoroorotic acid (5-

FOA). In this experiment, 2 of 12 yeast isolates lost 2 micron plasmid DNA (Figure 3-1).

This confirms our initial findings that pJPAll4 transformants lose 2 micron plasmids

with a sufficiently high frequency to allow pJPAll14 to be useful for curing a strain of the

2 micron plasmid.

Two Micron Circle Does Not Reduce Life Span

To determine if the presence of 2 micron plasmids influenced replicative life span,

microdissection-based life span determinations were done as described (86,92). The two

ciro strains, yAF7 and yAF8, obtained from the experiment presented in Figure I were

compared to the parental strain W303AR5. No apparent difference in replicative life

spans was observed (Figure 3-2). The average replicative life spans for yAF7, yAF8, and

W303AR5 were 22.7 (16.2), 21.9 (17.3), and 23.5 (15.9) generations, respectively.

Wilcoxon two-sample paired signed rank tests revealed no statistically significant

differences between the three life span curves (p<0.05). Thus, the presence of 2 micron










plasmids in W303AR5 does reduce replicative life span compared to two independently-

isolated, otherwise isogenic strains lacking 2 micron plasmids.


~-+- yA54FT -W-- yAF8 --&-- Wct303AR$













03 10 20 30 4

Generatoe


Figure 3-.iesa nlsso i n ir es.Nme fduhe el








Figre3-.Lfor ypAF7 alyAF8,fi and W303R, reaspetivly Thm e three curelsar



indistinguishable by Wilcoxon two-sample paired signed rank tests (p<0.05).

Two Micron Circle Does Not Accumulate in Old Cells

These findings indicate that 2 micron plasmids do not confer a disadvantage insofar

as replicative life span is concerned. This suggests that 2 micron plasmids do not

accumulate during the aging process. To test this prediction, ~6-generation old yeast

cells were prepared by magnetic sorting (see Materials and Methods) and 2 micron

plasmid DNA levels were analyzed by Southern blotting. No differences in 2 micron















~ Ib
IrZu ;B

~s~ B
~Wfig 5


Figure 3-3.Two micron plasmid levels in young and old cells. Old cells were isolated by
biotinylation and magnetic sorting (see Materials and Methods). Bud scars in
young and old cells were stained with Calcofluor and counted to determine
average age. DNAs from young and old cells were analyzed by Southern
blotting as described for Figure 3-1. Size markers (M) are shown (in kb). To
normalize levels of 2 micron plasmid DNA to genomic DNA levels, the blot
was stripped and rehybridized to 32P-labeled probe to URA3. 2 micron
plasmid and URA3 band intensities were quantitated, and no significant
difference was found between the ratios of 2 micron plasmid DNA to URA3
DNA in young and old cells.

plasmid DNA levels were observed between populations of cells with average ages of 1.1

and 6.2 generations (Figure 3-2). To normalize the amounts of 2 micron DNA present in

young and old cell samples to the amounts of genomic DNA present, the relative amount

of the URA3 gene was determined by Southern blotting (Figure 3-2). Quantitative

analysis of the intensities of bands corresponding to 2 micron plasmid DNA and URA3

was done and revealed no significant difference between normalized 2 micron DNA


~ud~ga P~~
l.f 6.2 M


2 C1~ rrrr ~arirr,










levels in young and old cells (data not shown). Previous studies have shown substantial

accumulation of ERCs (5- to 50-fold) and non-centromeric recombinant plasmids (5- to

25-fold) in 7-generation old yeast cells (86). Thus, 2 micron plasmids are unlike ERCs

and non-centromeric yeast plasmid vectors, and do not accumulate in old cells.

Summary

In chapter 2, 2 micron origin plasmids were shown to accumulate. The same is

not true for naturally occurring 2 micron circles. They do not accumulate; therefore, they

do not reduce lifespan. While only confirming the model of plasmid aging, the loss of 2

micron circles in transformants of pJPAll14 is very interesting. What is pJPAll14 doing

to cause 2 micron circle loss?















CHAPTER 4
A CELL'S LIMITED RESOURCES AND PLASMID COMPETITION

The main hypothesis being developed to explain ERC mediated aging revolves

around the idea that episomes cause a replication burden in cells. As cells age, they

accumulate episomes (93). The episomes eventually reach a high copy number. It is so

high, that it is about the same amount of DNA as the yeast genome (93). This means that

an old cell is replicating two or more times the amount of DNA it normally replicates.

This enormous amount of DNA could require all of a cell's replication factors and DNA

substrates to complete replication. If one of these factors or substrates is limiting, then

the cell may encounter problems during replication. This could result in mutations,

double strand breaks, etc. In order to further explore this idea, the following experiments

were completed.

Smllp, Meclp, RNR, and Rad53p Pathway

Smllp and Meclp are involved in a well know DNA damage and repair pathway

(94). Most importantly it senses replication fork slowing and stalls. The cascade starts

by Meclp sensing DNA damage or replication fork stalling. It then signals through

Rad53p to Smllp. Sm 11p, an inhibitor, releases and thereby activates Ribonucleotide

Reductase (RNR) (Figure 4-1). RNR makes dNTPs from NTPs by removing the 2'

hydroxyl from ribose. This reaction results in an increase of cellular pools of dNTPs and

the progression of stalled replication forks (94).









































Figure 4-1.Meclp, Rad53p, Smllp, and RNR pathway (94). This pathway ultimately
leads to the production of dNTPs.
SML1 Deletions Do Not Increase Life Span.
To see whether an increase in the cellular dNTPs pools would counteract the affects

of an episome's replication burden, a SM~L1 deletion was created with the insertion of

HIS3. By removing the RNR complex inhibitor, Smllp, dNTPs are continuously being


DCNA Jarrage,. rephla~tion blockE, & jstalled repli~tcano fbrks6










Trascrptina r





Rur p 1


RNR ~


gu


lation









produced no matter what the state of the cell. SM~L1 deletions are known to increase

dNTPs levels in the cell by 2-3 fold. In addition to looking at the life span ofSM~L1 and

sml1A strains, transformants of smllA were also test for an extended life span. The

plasmid used was pJPAll3. This is the ARS1 plasmid from Figure 2-1B and Table 2-1.

Transformed cells may help to amplify the affect sml1A has on a replication burden, since

they contain more episomes and have shorter life spans.


-*yAF10O+ARS
-=-smll n+ARS


yAF10
-*-sm/7AI


0 10 20 30 40
Generations


Figure 4-2.Life span ofSM~L1 deletions. yAF10 (HIS3; control) had a mean life span of
25.5 and a n=55. smllA had a mean life span of 25.9 and a n=57. yAF10 +
ARS (control) had a mean life span of 12.9 and a n=42. smllA + ARS had a
mean life span of 11.8 and a n=54. The ARS plasmid used was pJPAll3
(ARS 1).

The SM~Ll deletion had the same life span as the control (yAF 10). They were

statistically indistinguishable using the Wilcoxon two-sample paired signed rank test.









yAF 10 is the yAF5 strain with its his3 locus repaired to control for insertion of HIS3 into

snall. The transformed snlall cells also had the same life span as the transformed control

strain (Figure 4-2). Reproducing previous results, the pJPAll3 (ARS1) transformants

had a mean life span within the standard deviations of the life spans in Table 2-2.

Since replication is a doubling process, it is conceivable that an increase in dNTPs

of 2-3 fold is not enough to be seen on a life span. In other words, the life span assay

may not be sensitive enough. In old cells, episomes account for a large proportion of the

total DNA (could be more than half). Since they are retained due to mother cell bias, a

division occurring at an old age would increase the total DNA content by nearly double.

The only DNA being passed on to the daughter cell would be the chromosomes, not the

massively accumulated and newly replicated episomes. The key to this idea is that

episomes can reach a quantity larger than that of the genome. A snlall may only have

increased a cells life span by one doubling, not enough to be observed by life span.

Old Cells Do Not have an Increased Sensitivity to Hydroxyurea

Hydroxyurea (HU) is a chemical inhibitor of the RNR complex (94). It has been

widely used when studying Meclp and Smllp. Sn~all has an increased resistance to

HU. Strains that are snzllA neclA or sn~all rad53A have an increased sensitivity to

HU. Interestingly M~EC1 and RAD53 cannot be deleted without a SM~L1 deletion

suppressing their lethality (94). Smllp has a critical role in HU sensitivity because it is

the inhibitor of the RNR complex. Without SM~L1 it takes more HU to suppress the

larger pool of active RNR in the cell. IfM~EC1 or RAD53 are deleted then Smllp does

not release the RNR complex and the cell cannot make dNTPs. This is why sdl~A is

required when deleting M~EC1 and RAD53. M~eclA snal and rad53A sdl~A strains are

not less sensitive to HU because of Mec lp and Rad53p regulation of transcription factors










for the RNR proteins through Dunlp (Figure4-1). Without the activation of Dunlp, RNR

levels in the cell stay the same and cannot compensate for the inhibition by HU.

O mtM HUL 50 mMi HU 1008 mM HU 3200 mM~ HU




Young







Old





Figure 4-3.Hydroxyurea sensitivity of young and old cells from 0 mM to 200 mM. Each
plate has Hyve rows of pin stamps. Each row is a serial dilution of a strain.
The first row is a WT control yAF6. The second row is a WT control W1588-
4C. The third row is sdl~A. The fourth row is nzeclAsnlall. The fifth row is
rad53Asndl A.

A change in HU sensitivity by old cells would show that this DNA repair pathway

plays a role in the aging process. Two WT, sdl~A, nzeclA snlall, and rad53A sdl~A

strains were magnetically sorted to get young and old cells. Serial dilutions were pin

stamped onto minimal medium plates containing various levels of hydroxyurea. The first

pin stamping contained HU concentrations of 0, 50, 100, 200, 300, and 400 mM. As

expected the nzeclA snlall and rad53A sdl~A strains had an increased sensitivity to HU,

while the WT and snlall were more resistant. There were no noticeable differences in

the sensitivity of HU between the young and old cells (Figure 4-3). The 300 and 400 mM

HU concentrations are not shown because no strains were able to grow in those

conditions.









Om ML HU


10 mM HU


20 mMV HUI


Young


80Q mMr HU


4~0 mM HU


50 mM HU


Figure 4-4.Hydroxyurea sensitivity of young and old cells from 0 mM to 50 mM. Each
plate has Hyve rows of pin stamps. Each row is a serial dilution of a strain.
The first row is a WT control yAF6. The second row is a WT control W1588-
4C. The third row is sdl~A. The fourth row is nzeclAsnlall. The fifth row is
rad53Asndl A.










To further investigate HU sensitivity in old and young cells, a narrower range of

HU was examined for the second round of pin stamping. The concentrations of HU used

in this experiment were 0, 10, 20, 30, 40, and 50 mM. Again no distinction could be

drawn between the HU sensitivity of old cells to the HU sensitivity of young cells.

A closer look at concentrations between 0 to 10 mM and 100 to 300 mM may show

the differences we are looking for. The increase resistance to HU of smllA was not

shown; therefore, there needs to be a tighter range of concentrations between 100 and 300

mM. It is possible that old cells are more resistant to HU because they are up regulating

RNR.

Double Strand Breaks Do Not Increase in Old Cells

Replication slow zones are regions of DNA within the chromosomes at which

replication forks slow down. These zones were first discovered in M~EC1 mutants, where

the slow zones turned into double stranded breaks (DSB) (95). Since Meclp is the sensor

for stalled replication forks, M~EC1 mutants cannot fix stalled forks. In M~EC1 mutants,

replication forks spend more time at the slow zones. This leads to more DSBs in these

replication slow zones.

In old cells, the diminishing amounts of dNTPs caused by the episomal replication

burden could cause replication forks to stall more frequently and for longer periods of

time. This would in turn lead to more DSB. To test whether magnetically sorted old

cells have more DSB then young cells, pulse field gels were used to separate

chromosomes. After the gel was completed, it was transferred to positively charged

nylon and probed with the CHA1 probe. CHAl is located on one end chromosome III

(95). If a DSB occurred, then faint bands should appear below the chromosome III band.

These bands are shortened versions of chromosome III. No discernable difference could









be observed between young and old cells. There were no shorter bands on the blot;

therefore, there were no DSBs.

The original paper describing DSBs in nzecl mutants used a synchronized

population of cells. DNA was extracted from cells in the process of S phase. By using

cell synchronization, DSB formation in old cell might be able to be seen. Since there is

no positive control (nzeclA synchronized) in Figure 4-5, it is hard to say that the absence

of DSB detection conclusively shows that DSB do not form in old cells.










Figure~~,a 4-.oter fDBinyF W),W484C(TsdlnelsnlA n
ra53sdl. usefil gl a rn corin o atral admehos
Sothrnws raseredt psiiel cage nln ndralaln










Thgue pr5.othein tht ene DSB an sale repicaio forks in4 the) cella Mecaslpa,



phosphorylates o Rad53p (96). If t Rad53p pohryaion Olevel icess nodcelte



this pathway could be implemented in aging. After magnetically sorting old cells, protein

lysates were extracted. Several controls were also completed in parallel. HU causes a









signal cascade through this DNA repair pathway resulting in the phosphorylation of

Rad53p and the release of the RNR complex by Smllp. The phosphorylation can be seen

by a shift upward of Rad53p band on a gel (96). A rad53A smllA strain was added to

show which band is the Rad53p (the one not present in this strain). A meclA sml1A strain

was used to show a less phosphorylated Rad53p. The western blot shows that there was

no increase in the phosphrylation levels of Rad53p between young, old, and older (double

magnetic sort) cells. Even between cells that have have a variety of ERC levels (foblA,

WT, and sir2A), there was no difference in the phosphorylation of Rad53p.







Fiue -.Rd3ppophrlain ny un an ol el.Teupr ipre adi

Sinc thii a polyclona antbd, thr issm oseiicbnigt ad






CiuEN pl6.asmd53 ccanorlaio be seenan ld. Thesstanhvedfrntles ofpe ERsese band in tr
differetlie spans. 4 Sir2A prdcs the mhosthrlae ERs W rsoduc slghl less. Fh oblA

producs ver few pF3 is a stycoable pasmibd that cotins a ENelme oseicdnt. It repesnts


licens foring ofF3 ano single origin~ during the accumlatio ofEosf ERCs casea eioal





replication burden by abnormal licensing of ARSs throughout the genome, then pAF32

should fail to replicate more often in strains with more ERCs.













-* W303R5


11

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0


-m sir2A


-A- fob lA


0 10 20 30 40 50

Generations



Figure 4-7.Life span of W303R5 (WT), sir2A, and foblA during the CEN loss
experiment. W303R5 had a mean life span of 22.4 and an n = 29. Sir2A had
a mean life span of 12.6 and an n = 28. FoblA had a mean life span of 30. 1
and an n = 30.

We followed the inheritance pattern of pAF32 throughout the life span of the

various transformants. Because pAF32 contains the ADE2 gene, its inheritance can be

followed by colony color (Appendix I-K). After the pedigree analysis was completed,

the life spans of the three strains (with a CEN plasmid) were consistent with the life spans

that are already published in the literature (without a CEN plasmid) (Figure 4-7). After a

close analysis of the pedigrees, they show that the sir2A stain lost the plasmid at earlier

divisions than the both WT and foblA strains. WT, which has intermediate number of










ERC, had intermediate loss of pAF32. The foblA strain had the fewest ERCs and the

latest lost of the CEN plasmid (Figure 4-8). While there was a low n, this result suggests

that ERCs inhibit CEN plasmid replication. It also says that episomes may cause aging in

old cells by sequestering needed replication factors away from the genome.


-* W303R5


-m sir2A


-A- fob lA


1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0


10 20

Generation at which CEN Plasmid was lost


Figure 4-8.The age which WT, sir2A, and foblA lose plasmid. CEN plasmids were lost
at a mean generation time of 11.4, 6.25, and 14.3 (respectively). The number
of cells analyzed in each strain were n = 8, n = 4, and n = 7 (respectively).

Mitotic Stabilities in the Presence of ERCs

To further investigate ERC's role in the cell, mitotic stabilities were examined. A

change in mitotic stability of ARS plasmids may be expected in strains with various













levels of ERCs accumulation. FoblA, WT, and sir2A strains were tested with pAF31


(ARS), pAF32 (CEN), pJPAl33 (ARS), and pJPAl36 (CEN). The two sets of plasmids


were used to control for the use of the ADE2 or LEU2 selectable markers. The sir2A


strain had an increased mitotic stability of pAF31i. Normally pAF3 1 has a mitotic





n= 7 9 11 13 13 14


100


90 -


80 -


70 -


60 -


50 -


40 -


30 -


20 -


10 -


0-


-


-


-


-


-


-


-


-


-


W303
pAF31


foblA
pAF31


sir2A W303
pAF31 pAF32

Strain/Plasmid


foblA sir2A
pAF32 pAF32


Figure 4-9.Mitotic stabilities of pAF3 1 and pAF32. The two plasmids were tested in
strains with various amounts of ERCs.


stability of 16% (W303). In the sir2A strain, pAF3 1 had a mitotic stability of 29%. The


sir2A strain had a slight increase in the mitotic stability of pJPAl33, but it was not


significant. The mitotic stability went from 17% (W303) to 21% (sir2A), but these









numbers fell within the standard deviation of each other. This could be explained by the

smaller size of pJPAl33 from that of pAF3 1. Very small plasmids are known to have

different mitotic stability characteristics. The ARS plasmid, pAF31, may have reached a

critical threshold in size for ERCs to play a role in mitotic stability. The foblA strain's

inability to further reduce the mitotic stability of pAF31 shows that there may be a

critical mass of episomes a cell can handle. If the increase in ERCs from fob lA to W303

was not enough to change mitotic stability, then the decrease in size from pAF31 to

pJPAl33 could be not enough to change mitotic stability.


W303 foblA sir2A W303 fob1 sir2
pJPA133 pJPA133 pJPA133 pJPA136 pJPA136 pJPA136

Strain/Plasmid


Figure 4-10. Mitotic stabilities of pJPAl33 and pJPAl36. The two plasmids were
tested in strains with various amounts of ERCs.









Plasmid Accumulation in sir2A and foblA Strains

Again foblA, sir2A, and wt strains were transformed with pJPAl33 and pJPAl36.

Magnetic sorts were completed to obtain young and old cells. The episomal replication

burden model predicts that accumulating plasmid will compete. In this example, ERCs

will compete with pJPAl33 for the replication machinery. The accumulation of the ARS

plasmid will decrease in the strains with increased ERC production.











Fiue-1.S uathern ofsoigpasi opttonpeoeo. ldgse

genmi DNA yild a .3 k E2 ad the ad r lsi
derived.C













mostcopes411 Softher plasmid (29 plasmids/cll) iinoldcls pJPe l33 alon Xhad different


coy umbern i eecmpee n the youngti strains Toqatfhe copy numberswr6,46an2. wit res ett


























































I I -


foblA, WT, and sir2A. This again mimicked the results from the old cells, but to a lesser


degree. Young and old cells of all of the strains contained relatively the same number of


pJPAl36 (CEN). This argues for a competition between ARS episomes for replication


machinery in the cell. To further extrapolate this idea, episomes may also compete with


the genome for the replication machinery and cause aging. Another explanation of this


result may be that there is a critical mass of episomes allowed in the nucleus. After that


mass is reached episomes begin to be pushed out.


350 ,


300



-250



-


1 00 -


HYoung
SOld


50t-


sir2 plPA133


wt plPA133 fob1 plPA133 sir2 plPA136
Strains


wt plPA136 fob1 plPA136


Figure 4-12. Quantitation of plasmid competition Southern. Quantitaion was
completed on the blot in Figure 4-11 by a Phosphuor imager.

Summary

The mechanism by which episomes cause aging has been very illusive. In this


chapter several experiments have been designed to tease out the mechanism. Although


some experiments gave negative answers, they did give definitive answers. While the









exact mechanism of episomal aging is still not well known, the experiments strongly

suggest in what direction subsequent experiments should go.

The maj or experiments in this chapter show a few things:

*CEN plasmids are lost at younger generation times in strains with more ERCs.

*The mitotic stability of ARS plasmids increases in strains with more ERCs.

*Old cells of strains with more ERCs accumulate fewer ARS plasmids.

Together they say that as episomes accumulate there is an increasing strain on the

cells replication machinery.















CHAPTER 5
CHROMATIN SILENCINTG AND EPISOME FORMATION

The maj or role of Sirdp in aging is its ability to deacetylate histones. By

deacetylating histones in the rDNA, it is silencing the DNA. More specifically it is

compacting the chromatin and making it less accessible. This results in fewer ERCs

produced and in turn a longer life span. Strains that over express Sirdp have fewer ERCs

and live longer. Conversely SIR2 deletions create more ERCs and have a shorter life

span because it cannot deacetylate histones.

If the histone acetylation status in the rDNA is central in ERC aging, then limiting

a cells ability to acetylate histones could be as important as Sirdp ability to deacetylate

histones. Histone acetyl transferases (HATs) are the enzymes that acetylate histones. In

addition to the enzymes responsible for the acetylation and deacetylation of histones the

pool of acetyl CoA may be important to aging. Acetyl CoA is the substrate for HATs in

the acetylation of histones. A closer look at the production of acetyl CoA may lead to

mutants that increase life span. These ideas are illustrated in Figure 5-1.

Two enzymes are responsible for the production of acetyl CoA, Acslp and Acs2p

(Acetyl CoA Synthetase). Acslp has a Km 30 times lower than Acs2p (97). This does

not mean that it does most of the conversion of acetate to acetyl CoA. The two enzymes

are regulated very differently. Acslp is completely repressed by glucose (100 mg/L), up

regulated in ethanol, and further increased in acetate medium. Where as Acs2p is

maintained at a constant level in glucose and ethanol, but acts sporadically in acetate

(97). An ACS2 deletion cannot grow on glucose as a carbon source. This is because of






























At:


__ __ _~_ _


the repression of ACS1 by glucose. If ACS2 is deleted and ACS1 is repressed by glucose,

then there is insufficient ACS activity in the cell, and the cell cannot survive. The cell

needs acetyl CoA to survive.


-0,

















FDNA Stab~ilit


Are~tata


Lonlgevity
Agl ng
Figure 5-1. The acetylation of histones and its affect on ERC production and life span.

ACS2 and ACS1 is Required for Normal Life Span

Deletions ofACS1 and ACS2 were obtained from the Clusius lab. It was predicted

that deletions of the ACS genes would lead to an extended life span. This is because

acetyl CoA levels in the cell would drop. Histones would be acetylated less, and the

production of ERCs would decrease causing a longer life span.


EtH

Acetakiehyde


Acetyl CoA Co


s~4bc~HiStone~





rD~NA Inssta l4ilty










Life spans were completed on both glucose and ethanol. Surprisingly T23D, the

strain background, has a much longer life span than the W303R5 derivatives that have

been used in previous studies throughout this dissertation and other strains in published

papers. Never the less, there was a reduction in life span in acslA from the controls.

Interestingly acs2A had an even shorter life span than a~sclA (Figure 5-2). These

reductions in life span are opposite of what one might conclude, from the model

described in Figure 5-1.


-H-23D E -*-21 E
-K-T23D D -%-621 D


625 E


20 40 60
Generations


Figure 5-2.Life span of ACS deletions. T23D (control) on ethanol (E) had a mean life
span of 55.2 and an n=59. 621 (acslA) on ethanol (E) had a mean life span of
47.4 and an n=59. 625 (acs2A) on ethanol (E) had a mean life span of 16.6
and an n=51. T23D (control) on glucose (G) had a mean life span of 35.0 and
an n=59. 621 (acslA) on glucose (G) had a mean life span of 24.8 and an
n=59.









A closer look at metabolism shows that acetyl CoA is used for many processes in

the cell besides acetylation of histones. Acetyl CoA is one of the entrance points into the

Citric Acid Cycle. It is also used in lipid synthesis. ACS2 is known to be coregulated

with structural genes of fatty acid biosynthesis because of an upstream ICRE

(inositol/choline-response element). These and other pathways may be more important to

a cell's health than the predicted decrease in the production of ERCs by the ACS

deletions.

In addition to looking at the affects of Acslp and Acs2p in a cell, we inadvertently

observed a difference in life span between cells grown on glucose and cells grown on

ethanol as a carbon source. When T23D was grown on ethanol it had a mean life span of

55.2 and a maximum life span of 77 Generations. This is longer than the longest life

span we have seen published in the literature. To our knowledge the longest life span

published is a mean life span of 36. 1 and a maximum life span of 74 (98).

ACS2A Increases ERC Production

To further explore ACS1 and ACS2's role in aging, ERC production and

accumulation was measure in old and young cells of the ACS deletions. According to the

model illustrated in Figure 5-1, ACS deletions should lead to a decrease in ERC

production. Cells were grown in ethanol and then age fractionated by magnetic sorting.

T23D (control) had very few to no ERCs in the old cells. This may be why it has a

longer life span than W303R5. What is the genetic difference between T23D and

W303R5. T23D is a diploid strain while W303R5 is a haploid. Previous experiments

have shown that the ploidy of a strain is not important to life span (99). Another

difference is that W303R5 is ade2, his3, leu2, trpl, and ura3; and T23D has no







62


auxotrophic markers. Some of these genes can be compared by life spans that were

completely separately, and they do not seem to influence life span.

Interestingly, the ACS2 deletion produces more ERCs. While the ACS1 deletion

does not produce ERCs. Acs2A's ERC production may attribute to its shortened life

span.


9:
..I
IaIr ~


x:: x : :


Figure 5-3.Sorts of young and old ACS deletion strains. The southern blot was probe for
rDNA. Light banding in the old acs2A sample can be observed at a high
molecular weight. These are ERCs.

Summary

ACS1, ACS2, ethanol as a carbon source, and fatty acid biosynthesis are all linked

to aging. Further exploration is needed to fully understand how acetyl CoA is involved in









aging. Another noteworthy result is that T23D lives longer than W303R5. It would be

interesting to know what genetic differences contribute to its extended life span. Many

experiments could easily be designed to determine the genes that help it live longer.















CHAPTER 6
LOOKING AT POSSIBLE MECHANISM OF CELLULAR AGING

YCA1 and Apoptosis in Yeast

Apoptosis and aging are intimately linked. Since programmed cell death,

apoptosis, contributes to the aging of metazoans, looking at a known yeast caspase, an

apoptotic regulator, seemed reasonable. Yealp is the only known caspase like protein in

yeast (100). It has been shown to be required for hydrogen peroxide induced apoptosis.

When YCA1 is deleted, it increases yeast chronological life span (100). To further

investigate the role of Yealp in the aging process we constructed a YCA1 deletion in our

W303AR5 strain. It was created by using microhomology to YCA1 and inserting HIS3

inside of the gene (See Materials and Methods). After the insertion of HIS3 into YCA1,

we confirmed by southern that the constructed W303AR5 ycal::HIS3 strain was correct.

A control strain was also created by repairing the his3 locus of the W303AR5 strain.

This was to insure that the HIS3 status of the cell would not contribute to a lifespan

change. The two strains were compared through replicative life spans (Figure 6-1).

There was no statistical difference between the ycal deletion and the control strain. The

interpretation is that while ycal is necessary for a normal chronological life span, it is not

required for replicative life span. This experiment illustrates the fundamental difference

between a chronological and replicative life span. A chronological life span

demonstrates a cells resilience and ability to replicate after being in a saturated culture

full of cells and depleted of nutrients. In a replicative life span, cells are spaced out very

far from each other, so it is unlikely that they will run out of nutrients. It is a measure of









a cell replicative potential, not its resilience over time. It is a subtle difference, but

important when trying to address different questions about cellular aging.


-m Control


ycalAn


10 20 30 40
Generations


Figure 6-1.Life span of ycalA. This life span was completed on rich media (YPD). The
control strain had a mean life span of 22. 1 (n=60). The ycal deletion had a
mean life span of 21.4 (n=57).

Shu Gene Family and Mutation Suppression in Aging

Using a CAN1 forward-mutation assay, the SHU (sensitivity to hydroxyurea) genes

were discovered. These genes are required to prevent spontaneous mutations. In the

assay, 4,847 yeast deletion mutants (from the yeast deletion proj ect) were screened for

the ability to spontaneously become canavanine resistant (canl) (101).

In collaboration with the laboratory of Rodeny Rothstein, four of the genes

discovered in this screen (SHU1, SHU2, SHU3, and CSM~2) were looked at by replicative











-= W303R5 -* shu? A
-n- shu3n -m- csm2d


shu2n
4Xn


10 20 30 40

Generations


Figure 6-2. SHU genes role in life span. The mean life span of W303R5 is 24.5 (n=60).
The mean life span of shulA is 25.3 (n=60). The mean life span of shu2A is
25.8 (n=58). The mean life span of shu3A is 23.2 (n=56). The mean life span
of csm2A is 22.5 (n=59). The mean life span of the quadruple deletion (shulA,
shu2A, shu3A, and csm2A) is 21.5 (n=59).

life span. They are believed to be involved in DNA replication. These experiments were

completed in the Rothstein lab. These gene's ability to suppress mutations and their

involvement in DNA replication make them a candidate for having an increased life span.

The Rothstein lab had preliminary data that suggested this with one or more of the

mutants. After the experiment was completed all strains had very similar life spans

(Figure 6-2). A close look at the p-values obtained from the Wilcoxon two-sample paired









signed rank test shows that there may be a small difference between the strains (Table 6-

1). The experiment needs to be repeated (one of the few that has not been) before any

maj or conclusions can be drawn.

Table 6-1. P-values of the SHU deletion life spans.
Comparison #1 Comparison #2 P-Value

W303R5 shulA 0.032

W303R5 shu2A 0.003

W303R5 shu3A 1.22 x 10-7

W303R5 csm2A 9.07 x 10-6

W303R5 Quadruple a 4.50 x 10-s

shulA shu2A 0.090

shulA shu3A 1.14 x10-4

shulA csm2A 2. 12 x10-6

shulA Quadruple a 3.30 x 10-s

shu2A shu3A 1.47 x 10-4

shu2A csm2A 1.02 x 10-5

shu2A Quadruple a 4.23 x 10-6

shu3A csm2A 0.016

shu3A Quadruple a 1.68 x10-5

csm2A Quadruple a 0.002

Summary

This chapter focuses on ideas that were not specifically linked to our episomal

aging model. Because these concepts could have played a role in the broader scope of

aging, we explored them. It is now known that YCA1 does not affect replicative life span.






68


We also know that more experiments need to be completed to show definitively the

subtle difference between the SHU strains















CHAPTER 7
DISCUSSION

Budding yeast is an excellent system in which to study cell-autonomous

mechanisms of aging. Mechanisms linked to genome stability, metabolic damage, and

metabolic regulation have been found to regulate yeast replicative life span (45-

47,52,53,102). Sinclair and Guarente have proposed that a key regulator of life span is

the cellular level of extrachromosomal rDNA circles (ERC s) (54). To study this

proposal, we have used plasmids to model ERC inheritance and accumulation, two

processes that govern ERC levels in yeast cells. Our work shows that plasmid DNAs

bring about significant reductions in yeast life span. We Eind that ARS1~ and 2 CI origin

plasmids specifically accumulate in old yeast cells, and that the level of accumulation of

ARS1~ and 2 CI origin plasmids in old cells correlates with the extent of reduction in life

span. This is the first demonstration to our knowledge of an inverse relationship between

DNA episome level in old cells and reduction in life span. We Eind that plasmids have a

direct effect on life span and do not indirectly reduce life span by increasing

recombination at the rDNA locus and increasing ERC levels in transformed cells.

Analysis of the "terminal" morphology of senescent cells indicates that plasmids do not

cause a stochastic arrest in the cell cycle, which is consistent with a normal aging

process. Reduction in life span does not require that plasmids carry rDNA repeat

sequences, and the presence of a full-length, functional 9.1 kb rDNA repeat on a plasmid

does not augment reduction in life span. These Eindings confirm the work of Sinclair and

Guarente (54), and provide significant new support for their ERC model by directly









demonstrating a relationship between plasmid inheritance, plasmid accumulation, and

reduction in life span. Our studies also highlight the value of plasmids as tools to

investigate properties of ERC s that are relevant to the aging process in yeast.

Why Do ARS Plasmids Accumulate in Mother Cells?

It has long been appreciated that ARS plasmids are inherited asymmetrically and

accumulate in mother cells (43). This accounts for the high copy number and low mitotic

stability of ARS plasmids. However, accumulation of ARS plasmids in cells that are

multiple generations old has not been directly demonstrated. Our studies are the first to

directly demonstrate that ARS1-containing plasmids accumulate to high levels in old

yeast cells. Although ARS1 plasmid partitioning bias is well known, little is understood

about its underlying mechanism. One possibility is that plasmid partitioning bias is due

to the nature of cell and nuclear division in budding yeast. During closed mitosis in

yeast, an intact nucleus elongates along the axis of the mitotic spindle and adopts an

elongated "dumb-bell" shape due to constriction of the nucleus at the bud neck.

Chromosomes pass though the constriction at the bud neck by virtue of their attachment

to the mitotic spindle, which is able to exert force on chromosomes. In the absence of

spindle attachment, passage of DNA molecules through the constriction at the bud neck

may be limited. Consistent with this notion, the relatively small (1.45 kb) TRP RI

plasmid has been shown to be inherited efficiently and to exhibit high mitotic stability

(103). The small size of the TRP RI plasmid may allow it to readily distribute between

mother and daughter cells through the bud neck constriction. Commonly used yeast

recombinant DNA vectors are typically larger than the TRP RI plasmid and require cis-

acting sequences and trans-acting factors to be stably inherited.










Why Do Budding Yeast Exhibit a Mother Cell Plasmid Segregation Bias?

One possibility is that mother cell segregation bias is a mechanism to protect

progeny cells from potential "parasitic" effects of episomal DNAs acquired from the

environment. The 2 CI circle is a commensall" episomal DNA that Futcher has depicted

as a sexually transmitted selfish DNA (89). The 2 CI circle depends on its capacity to

overcome mother cell segregation bias (see below) in order to survive in a host

population in the absence of any selective value. Another possibility is that mother cell

segregation bias is a mechanism to increase the longevity of progeny cells by limiting

transmission of ERC s.

Why Do ARS1 Plasmids Bring About Cellular Senescence More Rapidly than Do
ERCs?

One possibility is that virgin mothers contain more plasmids than ERC s at the start

of life span experiments. Virgin mothers must contain at least one ARS plasmid, but

probably contain on average ~0.5 ERC per cell. The difference in origin strength

between ARS1~ and the rDNA ARS may also be important. ARS1 is a relatively "strong"

ARS, and capable of supporting rapid plasmid accumulation in mother cells. ERC s

contain a comparatively "weak" ARS that is likely to support only relatively slow

accumulation in mother cells. The rDNA ARS contains an ACS (ARS consensus

sequence) that departs from the consensus at position 1 a change that has been shown

to reduce ARS function, primarily by limiting DNA unwinding (81). This difference in

strength could explain why ARS1 plasmids bring about senescence in mother cells more

rapidly than do ERC s. ARS1~ plasmids are replicated more efficiently than ERC s, which

increases the rate of ARS1 plasmid accumulation in mother cells compared to ERCs.









Do Cis-acting Sequences that Counteract Mother Cell Segregation Bias Suppress
Reduction in Life Span by ARS1 Plasmids?

Yes, ARS1 CEN4 plasmids reduce life span to a lesser extent than ARS1 plasmids,

which is consistent with results of Sinclair and Guarente (54). However, inclusion of

CEN4 on ARS1~ plasmids suppresses the reduction in maximum life span by ARS1~

plasmids, but does not fully suppress the reduction in average life span. Our studies also

directly show that ARS1 CEN4 plasmids do not accumulate in ~7 generation old mother

cells. The reduction in life span is not specific for the combination ofARS1 and CEN4.

The combination ofARSH4 and CEN6 (in pRS314, (77)) reduces average life span with a

minimal effect on maximum life span. The fact that centromeric DNA elements suppress

reduction in maximum life span supports the conclusion that ARS1~ plasmids exert their

effect by accumulation in mother cells, as discussed above.

Do 2 Micron Circles Reduce Life Span?

It is initially surprising that ciro cells did not have an increased life span compared

to cir~ cells, especially since 2Cl origin plasmids accumulated in old cells. Quickly we

realized that the 2Cl circles and 2Cl origin plasmids were very different. 2Cl circles did not

accumulate in old cells and 2Cl origin plasmids did. With this knowledge of 2C

accumulation, it becomes obvious that 2Cl circles would not decrease life span.

Why Do 2 Micron Origin Plasmids Reduce Life Span?

Although both 2 CI origin plasmids and 2 CI circles contain the REP3 STB cis-acting

stability element, 2 CI origin plasmids contain a single 599 bp segment, whereas 2 CI

circles contain two 599 bp segments arranged as an inverted repeat (76,80). More

efficient autoregulation of 2 CI circle copy number and inheritance is likely to prevent

accumulation in old cells. It is important to note that 2 CI circles can be toxic to cells









when present at high copy number. Constitutive expression of the 2 CI amplification

machinery results in high copy number and has deleterious effects on cell growth (76).

Similarly, mutations in NIB1 ULP result in unusually high levels of 2 CI circles,

formation of large inviable or mitotically arrested cells, and clonal lethality (104).

Studies by Dobson and coworkers indicate that an abnormal form of Rep2p, a 2 CI circle-

encoded plasmid partitioning protein, accumulates in ulpl mutants, suggesting that ULP1

is involved in partitioning of 2 CI circles during mitosis (M. Dobson, personal

communication). This suggests that high levels of 2 CI circles in nib1 ulpl mutants may

result from asymmetric inheritance. In this sense, phenotypes associated with nibl/ulpl

defects may share mechanistic underpinnings with senescent phenotypes associated with

asymmetric inheritance of plasmids and ERC s.

Why Do 2 Micron Origin Plasmids Have an Intermediate Effect on Life Span?

Although 2Cl origin plasmids accumulate in ~7 generation old mother cells, they

attain levels approximately half that observed with ARS1~ plasmids. As mentioned above,

comparison of results with 2Cl origin and ARS1~ plasmids supports an important semi-

quantitative inverse relationship: the extent of plasmid accumulation in old cells

correlates with the extent of reduction in life span.

Why Does Transformation with pJPA114 Lead to 2 Micron Circle Loss?

Recent studies of 2 micron plasmid partitioning have made great strides in

revealing roles for cis-acting elements and trans-acting factors in substantial cellular and

molecular detail (105,106). These studies and earlier studies (76,87) suggest that

inheritance of 2 micron plasmids has little mechanistic overlap with inheritance of

replicating (ARS) plasmids such as pJPAll4. Thus, it seems unlikely that pJPAll4

competes with 2 micron circle for a limiting amount of (a) specific mitotic partitioning









factorss. Another possibility is that pJPAll4 adversely affects 2 micron circle copy

number, which in turn adversely affects transmission to daughter cells. We have

observed that 2 micron circle DNA levels are reduced 30-40% in ~?-generation old cells

containing yeast replicating plasmid pJPAll3, but not in ~?-generation control cells

lacking pJPAll3. pJPAll3 accumulates to high levels in ~?-generation old cells (86),

and perhaps 2 micron circle DNA levels are reduced as a result of this accumulation.

pJPAll4 attains a high copy number in young cells (86), and is likely to accumulate in

old cells, like pJPAll3. Although these Eindings are not conclusive, they are consistent

with competition between pJPAll4 and 2 micron circles for DNA replication factors

and/or precursors, which could lead to reduced 2 micron circle copy number and

impaired transmission to daughter cells.

By What Mechanism(s) Do Plasmids, and by Implication ERCs, Reduce Life Span
in Yeast?

It is clear that asymmetric inheritance of plasmid DNAs has the potential to burden

mother cells with high DNA content. If we assume that a 5 kb plasmid is replicated once

each S phase, and uniformly inherited by the mother cell during M phase, then 12

doublings will yield a plasmid DNA content in excess of the nuclear genomic DNA

content (5 X 212 = 20.5 Mb plasmid DNA content > ~13 Mb nuclear genomic DNA

content). Of course, this example is an oversimplification and omits factors such as

origin firing frequency and segregation efficiency. However, we note that after 12

generations, 90% of pJPAll13 (5.7 kb ARS1 plasmid) transformants were senescent and

after 20 generations, 90% of pJPAl33 (4.8 kb ARS1~ plasmid) transformants were

senescent. The fact that significant percentages of senescent mother cells arise between

10 and 20 generations is consistent with the accumulation of plasmid DNA content to a









level that approaches or exceeds nuclear genomic DNA content. Similarly, Sinclair and

Guarente have estimated that the ERC content of old cells exceeds the content of the

linear genome (54).

Why Are There Less Plasmid Accumulation in Strains that Produce More ERCs?

This concept of plasmid competition brings us to believe that there is a replication

burden in old cells. Two things could cause the loss of pAF32 (CEN), when it is in the

presence of ERCs. A limiting replication factor or DNA substrate could be soaked up by

the large quantity of ERCs and not allow the single copy pAF32 to replicate. In another

scenario the ERCs could act almost like a physical barrier making it harder for the

plasmid to leave the cell. Inheritance and replication are the two mechanisms that are

central to the characteristics and behavior of plasmids.

The mitotic stability in sir2A, WT, and foblA eliminates the concept of ERCs

acting as a physical barrier for inheritance of pAF32. It also raises new questions when

looking at the ARS plasmids (pAF31 and pJPAl33). Returning to the two plasmid

processes, ERCs could be increasing plasmid inheritance or replication. While it is

unlikely that ERCs are causing an increase in replication, a look at inheritance allows us

to start constructing some models. It is possible that there is a limited amount of space in

the nucleus. The total number of episomes cannot be higher than some critical mass of

DNA. This would cause the two accumulating episomes to be pushed out of cells and

inherited better. Saturation of the plasmid bias machinery could be another mechanism

that increases mitotic stability.

Episome accumulation in old cells of strains that produce various levels of ERCs

shows us that plasmid competition is very real. The reduced level of pJPAl33 in sir2A is

about half of the copy number than in W303R5. This is dramatic. The most important










part of the plasmid competition phenomenon is whether, it is an output of how episomes

causes aging. Mechanistically these two ideas could be very similar. If this is true, we

will be able utilize the versatility of plasmids to discover how ERCs cause aging.

Is There Episomal Aging in Metazoans?

While ERCs have not been found in metazoans, it is hard to say that the absence of

evidence for them proves there is no episomal aging is higher organisms. It is possible

that another highly repetitive sequence can recombine to form episomes, but they have

been very difficult to detect. Also, DNA viruses could be interpreted as episomes that

reach high copy within a cell. An analogy can be draw between ERC replication stress

and viral commandeering of a cell's replication machinery. Both may lead to problems

during DNA replication and hence genomic instability. Genomic instability could

facilitate the mutations and recombination in various cancer causing genes and increase

the incidence of cancer.















CHAPTER 8
MATERIALS AND METHODS

This chapter contains the methods and procedure used for experiments throughout

this dissertation.

Yeast Strains and Plasmids

W3 03AR5 (M4rATa leu2-3,11~2 his3-11,1~5 ura3-1 ade2-1 trpl-1 canl-100 RAD5

ADE2::rDNA, [cil-+], (54)) was obtained from D. A. Sinclair. yAF5 and yAF6 were

constructed by integrating linearized pRS305 and pRS306 (77), respectively, into the

leu2-3,11~2 or ura3-1 loci of W303AR5, respectively, and genotypes were confirmed by

Southern blotting. Plasmids were transformed into W303AR5 using a standard lithium

acetate method (107). All experiments were done with freshly-prepared, independently-

isolated, colony-purified transformants. Unless otherwise noted, yeast were grown on

selective SD "drop in" medium (88).

Descriptions of plasmids are provided in Table 1. A 200 bp fragment containing

ARS1~ was amplified by PCR with primers 5 '-GGAAGCTTCCAAATGATTTAGCATTATC-3 and

5'-CCGAATTCTGTGGAGACAAATGGTG-3' using template YRpl7. A 200 bp fragment

containing the rDNA ARS was amplified by PCR with primers

5 '-CCAAGCTTGTGGACAGAGGAAAAGG-3 and 5 '-GGGAATTCATAACAGGAAAGTAACATCC-3 using

template pJPA102 (rDNA repeat with Ahdl endpoints in pCR4, see below). A 753 bp

fragment containing CEN4 was amplified by PCR with primers

5 '-GCGGATCCCCTAGGTTATCTATGCTG-3 and 5 '-GGGAATTCCTAGGTACCTAAATCCTC-3 using

template YCp50. A 1346 bp region of 2 CI circle DNA, containing the REP3 STB cis-










acting stability element and a single 599 bp repeat region, was amplified by PCR with

primers 5 '-CCGGATCCAACGAAGCATCTGTGCTTC-3 and

5 '-CCAAGCTTTATGATCCAATATCAAAGG-3 using pRS424 as template. rDNA repeats were

amplified by PCR using as template size-selected (8-10 kb), genomic DNA that was

digested with the appropriate enzyme (Ahdl, Psil, or Xmal). The following primer pairs

were used: Ahdl endpointS, 5'-GGGATCCATGTCGGCGGCAGTATTG-3 and

5'-CCTGCAGiCTGTCCCACATACTAAATCTC~TTC-3'; Psil endpoints,

5'-GGGATCCTAATATACGATGAGGATGATAGTG-3' and

5'-CCTGCAGTAATAGATATATACAATACATGTT~TTTACC-3'; Xmal endpoints,

5 '-CCCGGGGCACCTGTCACTTTGG-3 and 5 '-CCCGGGTAAACCCAGTTCCTCACTAT-3' PCR was

performed for 20 cycles with 15-second denaturation and annealing times using PfuTurbo

DNA polymerase (Stratagene). PCR products were purified (Qiagen), digested with

restriction enzymes and ligated directly into recipient vectors, or cloned into pCR4-

TOPO (Invitrogen), excised, gel-purified, and ligated into recipient vectors (see Table 2-

1). ARS elements were cloned between HindIII and EcoRI sites. CEN4 was cloned

between EcoRI and BamHI sites. The 2 CI origin was cloned between HindIII and

BamHI sites. rDNA inserts were cloned between PstI and BamHI sites in pAFl15, which

is derived from pRS424 and contains loxP sites that were inserted at EcoRI and Spel sites

using annealed primer pairs: 5 -AAT`TATAAC TTCGTA TAA TGTAT`GC TATACGAAGTTA T-3' and

5'-AATTATAACTTCGTATAGCATACATTATACGAAGTTAT3 (EcoRI);
5'-CTAGATAACT~TCGTATAATGTATGCTATACGAAGTTA-3 and


5'-CTAGATAACTTCGTATAGCATACATTATACGAAGTTAT3 (Spel). All cloned inserts were

sequenced in their entirety. Plasmids pJPA105, pJPA106, and pJPA107 (that contain









rDNA inserts) were propagated in E. coli DH~a grown in LB media with 25 Clg/ml

carbenicillin at 300C to avoid insert instability.

Mitotic Stability

For each plasmid, five transformants were grown in selective SD liquid medium for

2 days at 300C to saturation (OD600 = 1.1-1.5; 0.5-1X107 ofu/ml; growth to late log gave

results similar to stationary phase). Approximately 200-250 colony forming units (cfu)

of each transformant were plated on non-selective SD medium, grown for 2-3 days at

300C, replica plated onto selective and non-selective agar media, and grown for 3-4 days

at 300C. After these plates grow the total number of colonies that grew under no

selection are counted from the first plate. The number of colonies that grew under

selection are counted from the second plate. Dividing the number of colonies from the

second plate by the number of colonies on the first plate gets the percent of cells in the

population that had the plasmid. This is the mitotic stability. It is simply the number of

colonies that contained the plasmid divided by the total number of colonies.

Replicative Life Span Determinations

Replicative life span determinations were done essentially as described (92) with a

few modifications. Six transformants were streaked individually on one side of an SD

agar plate, and 10 virgin mother cells from each (n=60) were positioned in an orthogonal

grid pattern. Virgin mothers that failed to give rise to 5 daughters were not included in

the data set. Due to the low mitotic stability of ARS-plasmids, it was necessary to start

with approximately 250 virgin mother cells from ARS1~-plasmid transformants to obtain

n=50-60 for life span determinations. A Zeiss Tetrad microscope equipped with 16X

eyepieces was used for micromanipulations as described (88). SD agar plates were

weighed at the beginning of each experiment and sterile water was pipetted into four









small notches at the edge of each plate on a daily basis to compensate for evaporation and

prevent increases in osmolality, which could potentially affect results (108). During life

span experiments, plates were incubated at 300C during the daytime and stored overnight

(~12 hours) at 140C. We found that extended periods (>24 hours) at 140C reduced life

spans of transformants and control strains (data not shown). At the end of a life span

experiment, mother cells not having divided for 2 days were transferred to non-selective

SD or YPD medium, and cells that resumed mitosis were excluded from the data set.

This allowed us to exclude data from mother cells that stopped dividing due to plasmid

loss rather than due to cell senescence. Data were entered into an Excel spreadsheet

template file (available on request) that automatically calculated relevant life span data

values and performed Wilcoxon two-sample paired signed rank tests. Images of terminal

cells were collected using a Spot-2 CCD camera (Diagnostic Imaging) affixed to a Zeiss

Tetrad microscope and "terminal" cell morphology analysis was done as described (70).

Southern Blot Analysis and Quantitation

DNA was extracted from yeast cells using a glass beads/phenol method, digested

with restriction enzymes according to the supplier (New England Biolabs), separated on

0.8% agarose gels (200 V/hours), and capillary transferred to positively-charged nylon

membrane under alkaline conditions using standard methods (109). For each plasmid

copy number and ERC monomer level determination, five plasmid transformants were

analyzed in parallel. Digestion with BamHI or PstI yielded single plasmid-specific or

genome-specific bands of different sizes that hybridized tO 32P-labeled probe generated

by random-primed labeling (New England Biolabs). PstI and BamHI do not cleave

rDNA. Genomic bands were used as internal standards for measurements of plasmid

levels. Chromosomal rDNA bands were used as internal standards for measurements of









ERC monomer levels. Blots were hybridized first to GRA3 or LEU2 probe, followed by

stripping and hybridization to rDNA probe. Data from the same blots were used to

prepare Figures 2 and 3B. Southern data were acquired with a Typhoon PhosphorImager

and analyzed using ImageQuant software (Molecular Dynamics).

Pulse field gel electrophoresis was completed at 14oC with a Bio-Rad CHEF-DR II.

1% agarose gels were run in 0.5X TBE for 30 hours. The voltage used was 200V with a

switch time starting at 5 seconds and ending at 30 seconds.

Magnetic Cell Sorting

At any given time 1 in 512 cells is an eight generation old cell. This is due to the

nature of a doubling population. 1/2 of the cells are new, zero generation daughters. 1/2

of the cell remaining (1/4) are 1 generation old cells. 1/2 of the cell remaining (1/8) are 2

generation old cells. This continues until the number becomes increasingly smaller. To

extract the small number of old cells from a large population of cells, magnetic sorting is

used. 1x10" cells are grown up and labeled with biotin. They are then grown over night

in 1 liter of liquid medium. Since new cell wall synthesis occurs at the bud of the

emerging cell, no biotin is transferred to the newly divided daughters. This results in a

large population of cells that have their oldest cells labeled with biotin and the young

cells are not. The cells are then spun down and concentrated into a smaller volume.

Strep-avidin coated magnetic beads are mixed with the cells for 2 hours at 4oC. All of the

subsequent steps are done in the cold to ensure that the cell do not continue to grow. The

strong interaction between biotin and avidin allows the magnetic beads to bind to the old

cells. The old cells are then pulled out of solution with a strong magnet and the young

cells are washed away. Eight washes are used to ensure that the population acquired at









the end of the experiment is homogeneously old. The old cell final product is ready for

further use in other experiments.

Budscar Histograms

After a sort for old cells, a bud scar histogram is conducted to determine the age

distribution of the cells collected. When a cell divides a bud scar ring is formed at the

point of budding and separation. The bud scar can be stained with a fluorescent dye

calcofluor white MR2. A small fraction of old and young cells are stained with

calcofluor and the number of bud scars on 50 cells are recorded. This creates a histogram

of the number of cells vs. the number of budscars.

rDNA Recombination Assay

The rDNA recombination assay is designed to quantitate the level of recombination

at the rDNA locus. W303AR5 has an ADE2 gene within the rDNA locus. In the absence

of the ADE2 gene colonies become red in color, while wild type ADE2 are white. This

color phenotype allows the scoring of ade2 colonies to be very easy. Saturated liquid

cultures were prepared from five transformants. They were diluted and spread on 15 cm

selective SD agar plates containing 5 Clg/ml adenine hemisulfate and 5 Clg/ml histidine to

enhance red color production. They are allowed to grow at 30oC for 2-3 days. The

plates are then placed at 4oC for 1-2 days for the color to develop. The number of half

sector colonies, colonies that are half red half white, are scored in comparison to the

number of all white colonies. All other partially sectored or red colonies are ignored.

The reason only half sector colonies are scored is because half sectored colonies are

colonies that have lost the ADE2 marker on the first division in the colony formation. A

completely red colony may have become red at any time in growing in the liquid culture.






83


By looking at the first division to forming a colony, all of the data can be normalized to a

single mitotic event.















APPENDIX A
STRAIN: W303AR5+pJPAll3 MEDIA: SD aHLW

15 20 25 30 35 40 45 50 55 60


8 15 10 7 17 11 16 21 6 11


85 8 1120


20 7 6


18 10 17 14 6 15 21 16 20


8 12 16 15 9 18 10 22 24


?5 21


g ;16 8


18 21 13 19 93 8 10 24 10 6

Terminal morphology of senescent cells with its life span directly below the image.















APPENDIX B
STRAIN: W303AR5+pJPAll6 MEDIA: SD aHLW

15 20 26 30 35 40 45 50 55 60


6 21


12 28


31 24


30 21 27


7 20 33L 30


11 38


18 18


915 18 25


B 9s 5 32


22 32 25


10 34 29 29 28


32 12 29 27' 9


19 27 21 37 24


Terminal morphology of senescent cells with its life span directly below the image.















APPENDIX C
STRAIN: W303AR5+pJPAl38 MEDIA: SD aHLW

15 20 26 30 35 40 45 50 55 60


10 13 15 9


6 10 14 6


19 10


7 7 12 9 16


23 26


29 11 9


23 19


5 15 28


29 17 16; 5


31 11


12 12 24 15 23


Terminal morphology of senescent cells with its life span directly below the image.















APPENDIX D
STRAIN: yAF6 IVEDIA: SD aHLW

15 20 26 30 35 40 45 50 55 60


29


29 32 30 23 30 27 25 33 22


24 29 34 33 35 26 35 24 32 33


24 34 32 21 38 28


37 42 3r;


37 29 33 30 31 19


38 35 38 41


23 32 42


25 31 34 38 35 31 38 33 29

Terminal morphology of senescent cells with its life span directly below the image.















APPENDIX E
STRAIN: W303AR5+pJPAl33 MEDIA: SD aHWu

15 20 26 30 35 40 45 50 55 60


9 11 14 10


10i 7


12 12 5 17 10 5 10


Terminal morphology of senescent cells with its life span directly below the image.




Full Text

PAGE 1

BUILDING AN EPISOMAL MODEL OF AGING IN Saccharomyces cerevisiae By ALARIC ANTONIO FALCN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Alaric Antonio Falcn

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This document is dedicated to Peri A. Tong, Manuel A. Falcn, and Beverly L. Metcalfe for their unwavering support.

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iv ACKNOWLEDGMENTS I thank my mentor, John P. Aris, and my committee (William A. Dunn, Thomas C. Rowe, and Brian Burke) for helping me become a scientist.

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v TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi CHAPTER 1 BACKGROUND AND SIGNIFICANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Sir2p, rDNA, and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Extrachromosomal rDNA Circles are Discovered . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Components of the rDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Fob1p and its Role in ERC Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 ARS of the rDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Asymmetric Inheritance of ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2 DEFINING THE LINK BETWEEN EPISOMES AND AGING . . . . . . . . . . . . . . . . 9 Roles of Different Cis-Acting Plasmid Sequences in Reduction of Yeast Replicative Life Span . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Plasmid Inheritance Correlates with Reduction in Yeast Life Span . . . . . . . . . . . . 17 Plasmids Do Not Significantly Increase ERC Levels . . . . . . . . . . . . . . . . . . . . . . . 20 Plasmid Accumulation Correlates with Reduction in Life Span . . . . . . . . . . . . . . . 22 Terminal Cell Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Do Functional rDNA Transcriptional Units Play a Role in Reduction in Life Span? 31 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3 TWO MICRON CIRCLE: A NATURALLY OCCURRING EPISOMES ROLE IN AGING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 New Method for Removal of Two Micron Circle . . . . . . . . . . . . . . . . . . . . . . . . . 36 Two Micron Circle Does Not Reduce Life Span . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Two Micron Circle Does Not Accumulate in Old Cells . . . . . . . . . . . . . . . . . . . . . 39 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

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vi 4 A CELLS LIMITED RESOURCES AND PLASMID COMPETITION . . . . . . . . 42 Sml1p, Mec1p, RNR, and Rad53p Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 SML1 Deletions Do Not Increase Life Span. . . . . . . . . . . . . . . . . . . . . . . . . . 43 Old Cells Do Not have an Increased Sensitivity to Hydroxyurea . . . . . . . . . . . 45 Double Strand Breaks Do Not Increase in Old Cells . . . . . . . . . . . . . . . . . . . . 48 Phosphorylation of Rad53p Does Not Increase in Old Cells . . . . . . . . . . . . . . 49 ERC Competition with a CEN Plasmid Throughout Yeast Life Span . . . . . . . . . . . 50 Mitotic Stabilities in the Presence of ERCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Plasmid Accumulation in sir2 and fob1 Strains . . . . . . . . . . . . . . . . . . . . . . . . . 55 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 5 CHROMATIN SILENCING AND EPISOME FORMATION . . . . . . . . . . . . . . . . 58 ACS2 and ACS1 is Required for Normal Life Span . . . . . . . . . . . . . . . . . . . . . . . . 59 ACS2 Increases ERC Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 6 LOOKING AT POSSIBLE MECHANISM OF CELLULAR AGING . . . . . . . . . . 64 YCA1 and Apoptosis in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Shu Gene Family and Mutation Suppression in Aging . . . . . . . . . . . . . . . . . . . . . . 65 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 7 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Why Do ARS Plasmids Accumulate in Mother Cells? . . . . . . . . . . . . . . . . . . . . . . 70 Why Do Budding Yeast Exhibit a Mother Cell Plasmid Segregation Bias? . . . . . . 71 Why Do ARS1 Plasmids Bring About Cellular Senescence More Rapidly than Do E R C s? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Do Cis -acting Sequences that Counteract Mother Cell Segregation Bias Suppress Reduction in Life Span by ARS1 Plasmids? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Do 2 Micron Circles Reduce Life Span? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Why Do 2 Micron Origin Plasmids Reduce Life Span? . . . . . . . . . . . . . . . . . . . . . 72 Why Do 2 Micron Origin Plasmids Have an Intermediate Effect on Life Span? . . . 73 Why Does Transformation with pJPA114 Lead to 2 Micron Circle Loss? . . . . . . . 73 By What Mechanism(s) Do Plasmids, and by Implication E R C s, Reduce Life Span in Yeast? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Why Are There Less Plasmid Accumulation in Strains that Produce More ERCs? . 75 Is There Episomal Aging in Metazoans? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 8 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Yeast Strains and Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Mitotic Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Replicative Life Span Determinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Southern Blot Analysis and Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

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vii Magnetic Cell Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Budscar Histograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 rDNA Recombination Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 APPENDIX A STRAIN: W303AR5+pJPA113 MEDIA: SD aHLW . . . . . . . . . . . . . . . . . . . . . . . 84 B STRAIN: W303AR5+pJPA116 MEDIA: SD aHLW . . . . . . . . . . . . . . . . . . . . . . . 85 C STRAIN: W303AR5+pJPA138 MEDIA: SD aHLW . . . . . . . . . . . . . . . . . . . . . . . 86 D STRAIN: yAF6 MEDIA: SD aHLW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 E STRAIN: W303AR5+pJPA133 MEDIA: SD aHWu . . . . . . . . . . . . . . . . . . . . . . . 88 F STRAIN: W303AR5+pJPA136 MEDIA: SD aHWu . . . . . . . . . . . . . . . . . . . . . . . 89 G STRAIN: W303AR5+pJPA148 MEDIA: SD aHWu . . . . . . . . . . . . . . . . . . . . . . . 90 H STRAIN: yAF5 MEDIA: SD aHWu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 I STRAIN: W303R5 +pAF32 MEDIA: YPD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 J STRAIN: FOB1 +pAF32 MEDIA: YPD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 K STRAIN: SIR2 +pAF32 MEDIA: YPD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 L PLASMIDS USED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 M STRAINS USED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

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viii LIST OF TABLES Table page 2-1. Plasmids used in this study. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2-2. Life span data summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 6-1. P-values of the SHU deletion life spans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 L-1. The plasmids used throughout this dissertation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 M-1. The strains used throughout this dissertation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

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ix LIST OF FIGURES Figure page 1-1. The pseudoERC strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1-2. The rDNA repeat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1-3. Fob1 mediated expansion of the rDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1-4. ERC Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2-1. Life span analysis of plasmid-transformed yeast . . . . . . . . . . . . . . . . . . . . . . . . . 15 2-2. Plasmid inheritance studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2-3. Extrachromosomal rDNA circle ( E R C ) formation in yeast transformants . . . . . . 22 2-4. Plasmid DNA and extrachromosomal rDNA circle ( E R C ) levels in young and old cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2-5. Plasmid DNA and extrachromosomal rDNA circle ( E R C ) levels in young and old cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2-6. Terminal morphology of senescent cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2-7. Life span analysis of yeast transformed with plasmids containing rDNA repeats. 32 3-1. Southern blot analysis of pJPA114 transformants . . . . . . . . . . . . . . . . . . . . . . . . 37 3-2. Life span analysis of cir + and cir 0 yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3-3. Two micron plasmid levels in young and old cells . . . . . . . . . . . . . . . . . . . . . . . . 40 4-1. Mec1p, Rad53p, Sml1p, and RNR pathway (94). . . . . . . . . . . . . . . . . . . . . . . . . 43 4-2. Life span of SML1 deletions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 4-3. Hydroxyurea sensitivity of young and old cells from 0 mM to 200 mM . . . . . . . 46 4-4. Hydroxyurea sensitivity of young and old cells from 0 mM to 50 mM. . . . . . . . . 47

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x 4-5. Southern of DSB in yAF6 (WT), W1488-4C (WT), sml1 mec1 sml1 and rad53 sml1 .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4-6. Rad53p phosphorylation in young and old cells . . . . . . . . . . . . . . . . . . . . . . . . . . 50 4-7. Life span of W303R5 (WT), sir2 and fob1 during the CEN loss experiment. . 51 4-8. The age which WT, sir2 and fob1 lose plasmid . . . . . . . . . . . . . . . . . . . . . . . 52 4-9. Mitotic stabilities of pAF31 and pAF32 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4-10. Mitotic stabilities of pJPA133 and pJPA136. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 4-11. Southern of showing plasmid competition phenomenon.. . . . . . . . . . . . . . . . . . . 55 4-12. Quantitation of plasmid competition Southern . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 5-1. The acetylation of histones and its affect on ERC production and life span. . . . . . 59 5-2. Life span of ACS deletions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 5-3. Sorts of young and old ACS deletion strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 6-1. Life span of yca1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 6-2. SHU genes role in life span.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

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xi 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 BUILDING AN EPISOMAL MODEL OF AGING IN Saccharomyces cerevisiae By Alaric Antonio Falcn May, 2004 Chair: John P. Aris Major Department: Anatomy and Cell Biology Aging in Saccharomyces cerevisiae is under the control of multiple pathways. The production and accumulation of extrachromosomal rDNA circles ( E R C s) is one pathway that has been proposed to bring about aging in yeast. To test this proposal, we developed a plasmid-based model system to study the role of DNA episomes in reduction of yeast life span. Recombinant plasmids containing different replication origins, cis -acting partitioning elements, and selectable marker genes were constructed and analyzed for their effects on yeast replicative life span. Plasmids containing the ARS1 replication origin reduce life span to the greatest extent of the plasmids analyzed. This reduction in life span is partially suppressed by a CEN4 centromeric element on ARS1 plasmids. Plasmids containing a replication origin from the endogenous yeast 2 micron circle also reduce life span, but to a lesser extent than ARS1 plasmids. Consistent with this, ARS1 and 2 micron origin plasmids accumulate in ~7-generation-old cells, but ARS1/CEN4 plasmids do not. Importantly, ARS1 plasmids accumulate to higher levels in old cells than 2 micron origin plasmids, suggesting a correlation between plasmid accumulation

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xii and life span reduction. Reduction in life span is not an indirect effect of increased E R C levels, nor the result of stochastic cessation of growth. The presence of a fully functional 9.1 kb rDNA repeat on plasmids is not required for, and does not augment, reduction in life span. These findings support the view that accumulation of DNA episomes, including E R C s, cause cell senescence in yeast. The endogenous 2 micron circle is a naturally occurring episomal DNA. Loss of the 2 micron circle can be facilitated with the transformation of an ARS containing plasmid. Since 2 micron circles are episomes, and episomes can cause aging, experiments were complete to show that it does not accumulate in old cells and does not cause aging. In strains that contain more ERCs, ARS plasmids do not accumulate as much. There is an episomal competition phenomenon. While it is not known what the episomes are competing for, it can be demonstrated that as the number of different episomes increase the rate of accumulation for each episome decreases.

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1 CHAPTER 1 BACKGROUND AND SIGNIFICANCE Saccharomyces cerevisiae is a single-ce lled, budding yeast. During mitosis, budding yeast divide asymmetrically. This is different from fission yeast where the two cells produced by mitosis are indistinguishable. With budding yeast, the new smaller cell (the daughter cell) emerges from the older, larger cell (the mother cell) (1-4). Because the mother cell can be distinguished from its daughters, a mother can be followed throughout all of its divisions. As mothers age, they become enlarged, their cell cycle slows, and they become sterile (2,5). Daughters can be physically separated from their mothers with a microdisection microscope. Physical separation is necessary, because it is difficult to follow a mother cell through all of its divisions if it is obscured by daughter cells and daughters of daughter cells. By removing the daughters from mothers, and simultaneously tallying the number of divisions a mother completes, the cells replicative life span can be determined. The replicative life span is defined as the number of divisions a mother cell completes (6). With the ability to conduct replicative life span experiments and the ease of genetic manipulation, Saccharomyces cerevisia e is an excellent model organism for aging studies. Sir2p, rDNA, and Aging Long and short lived aging mutants have been isolated in Saccharomyces cerevisiae (7-10). One of the first such mutants identified was SIR4-42 a long lived mutant that resulted in the localization of the SIR complex to the nucleolus (3,8). The silent information regulator (SIR) complex is involved in the silencing of chromatin at

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2 the telomeres, mating type loci, and rDNA (2,11,12). The rDNA serves as a nucleolar organizing region in eukaryotic cells, and is the site of transcription of pre-rRNA (11). In addition to the findings that implicated the rDNA in life span, the nucleolus was found to be enlarged and fragmented in old yeast cells (2,3). This was consistent with the notion that the rDNA played a role in life span determination. More recently, the silencing protein Sir2p was found to be a nucleolar protein specifically involved in silencing at the rDNA locus (3). Loss of function sir2 mutations reduce life span (3,12), whereas SIR2 overexpression extends life span (13). Sir2p is now known to function as a histone deactylase that plays a central role in modulating chromatin structure (13,14). It has been tied to the extension of life span in metazoan organisms (15) as well as being linked to the caloric restriction model of aging (16). Extrachromosomal rDNA Circles are Discovered Based on SIR4-42 and other findings, Sinclair and Guarente in 1997 showed that old mother cells accumulated extrachromosomal rDNA circles (ERCs). This was proven by the use of 2D chloroquine gels and by Southern blots for rDNA. 2D chloroquine gels are used to look for closed circular DNA molecules (17-19). Old cell undigested DNA on 2D chloroquine gels showed rDNA episomes. The old cells had rDNA episomes and young cells did not (20) To test the role of ERCs in yeast mother cell aging, replicative life spans were conducted on cells that had been given pseudoERCs. PseudoERCs are induced by using a plasmid with a partial rDNA repeat flanked by loxP sites and a plasmid with Cre recombinase under the control of the galactose promoter. On adding galactose, the galactose promoter allows the expression of Cre recombinase (21). Cre recombinase then recombines the sequence between the loxP sites out of the plasmid (22). This creates the pseudoERC, a partial rDNA repeat with a selectable marker and no

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3 extraneous segregation or replication mechanisms (Figure 1-1). These pseudoERCs cause an earlier onset of senescence (20). More specifically, the cell that had the induction of pseudoERCs had a lower replicative life span (20). This suggested that ERCs are a cause of aging, and are not produced as an effect of aging. This experiment is the central proof of the ERC mediated aging model. The finding that ERCs could cause aging was a completely novel aging mechanism. Figure 1-1. The pseudoERC strategy. Activation of the GAL1 promoter by galactose produces Cre recombinase. Cre mediates recombination at the loxP sites resulting in the excision of the ARS CEN and the creation of a pseudoERC (20). Components of the rDNA The ribosomal DNA (rDNA) is present on chromosome XII in a single linear array of between 100 and 200 head-to-tail repeats (2,3,11,23). Each 9.1 kb rDNA repeat is responsible for producing the 5S and 35S pre-rRNA (Figure 1-2) (7,24) These RNAs are later processed and packaged with proteins to form a ribosome (24,25). One half of rDNA repeats are usually silent and not actively transcribing the rRNA (11,26). This excess capacity is to ensure that the vital function of protein synthesis will not be hindered by the lack of rRNA.

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4 Figure 1-2. The rDNA repeat. 5S and 35S pre-rRNA are both transcribed in the rDNA. The 35S is later processed into 18S, 5.8S, and 25S rRNA. Within the NTSs are the RFB and ARS-rD (both have been shown to be essential for ERC formation and replication, respectively). Fob1p and its Role in ERC Production The protein Fob1p is required for the replication fork block (RFB) in the nontranscribed spacer 1 (NTS1) (23,27) It is implicated in the formation of ERCs (7). The RFB blocks one of the replication forks within the replication bubble (23). This makes replication unidirectional, in the direction of 35S pre-rRNA transcription (23) The RFB has been observed in yeast, frog, mouse, human, and plant (28-32). The ultimate function of the RFB site in yeast is to allow the cell to expand and contract the number of repeats in the rDNA array by homologous recombination (23) (Figure 1-3). Because recombination is initiated at a DNA double stranded break (DSB), a crossover may occur within a sister chromatid by the formation of a Holliday structure (23). Conversely, recombination can occur upstream, resulting in the loss of a repeat (23). An apparently unintended consequence of RFB function is the increased production of ERCs (Figure 1-4) (7) Holliday structures within the rDNA occur 3.6 times per cell cycle (33), indicating rampant recombination at the locus. In FOB1 deleted strains, there is reduced recombination at the rDNA locus, because there is no RFB at the RFB site (34). This

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5 reflects fob1 s reduced formation of ERCs and longer life span (7). This further implicates ERCs in the aging process. Figure 1-3. Fob1 mediated expansion of the rDNA. (A) Fob1p acts at each RFB site in rDNA. (B) Replication begins at ARS-rD and two replication forks travel in opposite directions. (C) The replication fork traveling in the opposite direction of 35S transcription is stopped at RFB site. The other replication fork continues. A double stranded break can occur at the RFB site. (D) A Holliday structure forms and homologous recombination with the sister chromatid can repair the break. (E) The closest replication bubble catches up to the recombination site creating two separate strands of DNA, one of which has an additional repeat.

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6 Figure 1-4. ERC Formation (A) An ERC can be formed by homologous recombination and the looping out a circular DNA. (B) They can also be formed by recombination of the free end of DNA, the product of Fob1p RFB (27). ARS of the rDNA Within every rDNA repeat, there is an autonomously replicating sequence (ARS) or origin of replication (35,36) ARSs are AT rich sequences of DNA, to which the origin recognition complex (ORC) binds (37,38). The ORC complex of proteins is essential for the initiation of DNA replication (37,38). The ARS-rDs (rDNAs ARS) biological function is as a site for the initiation of DNA replication within the rDNA repeat. It is necessary because the repeat locus consists of one to two hundred headtotail 9.1 kb repeats (approximately 1,500 kb in length) (39). Normally, ARSs are spaced approximately 40 kb apart throughout the yeast genome (37,39,40). By putting an ARS in every repeat, replication of the genome through this lengthy region can occur more efficiently than with an ARS at either end of the locus (39). The ARS will also allow episomal rDNA, such as ERCs, to replicate (20,35) The ARS-rD is considered a weak ARS. That is, any given ARS-rD fires less than once per cell cycle (35,36,39) Because ARS-rD occurs once in every repeat, every third ARS can fire and still replicate the locus effectively. The distance between firing ARSs is approximately 30 kb. The

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7 activity of the ARS-rD has been linked to transcriptionally active 35S genes (which were in turn linked to nonsilent euchromatic regions of the locus) (41,42). Asymmetric Inheritance of ERCs A phenomenon associated with ERCs is their asymmetric segregation. There is a natural tendency for the ERCs and ARS plasmids to stay within the mother cell during cell division (2,3,43) This was demonstrated by pedigree analysis, a technique that follows the segregation of a non-Mendelian trait through mitosis (20). Mother cells have a bias to retain the plasmids and not pass them on to their daughters (43) Although this phenomenon was discovered in 1983, little is known about the mechanism that retains the plasmids in the mother cells. With ERCs being excised from the genome, replicated by their endogenous ARS, and segregated preferentially to the mother; there is a massive accumulation of the episomes in older cells. This is the model of ERC mediated aging (20). Although the amount of ERCs in very old cells is not known, the number estimated to be in cells after 15 generations is 500 to 1000 ERCs (20). This accumulation is thought to be behind the mechanism of ERC mediated aging (20). Summary A major tenet of the ERC mediated model for replicative aging in Saccharomyces cerevisiae is that ERCs are nothing more than episomal DNA molecules with an ARS (20). Evidence for this comes from the observation that a yeast shuttle vector, containing only an ARS, reduced replicative life span (compared to a control plasmid containing an ARS and a centromeric (CEN) element) (20). CEN plasmids are maintained at low copy number and segregate with high fidelity to daughter cells just like chromosomes (44) ARS plasmids attain a high copy number and show a bias toward retention in mother cells during mitosis (similar to ERCs) (43) The fact that the ARS plasmid can shorten

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8 yeast mother cell life span suggests that the rDNA sequence per se does not contribute to ERC mediated aging, and that potentially any extrachromosomal DNA able to replicate may reduce life span (20).

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9 CHAPTER 2 DEFINING THE LINK BETWEEN EPISOMES AND AGING The yeast Saccharomyces cerevisiae has proved to be a valuable model organism for investigating mechanisms of cellular aging (45-47). Central to the biology of aging in S. cerevisiae is an asymmetric cell division process that gives rise to mother and daughter cells with different characteristics. Mother cells have a limited capacity to produce daughter cells, and the decline in this capacity with each generation is referred to as replicative aging. The limited replicative potential of yeast mother cells has been recognized since the 1950s (48). Pioneering studies in the Jazwinski and Guarente laboratories postulated the existence of a senescence factor/substance that accumulates in mother cells and is transmissible to daughters (49,50). Work in the Guarente lab identified a heritable "age" locus that regulates yeast life span (51). More recent studies have made clear that allelic variation at single genetic loci can markedly affect yeast life span, including extension of life span. This indicates that a process as complex as cellular aging is controlled by a hierarchical regulatory system. Like in other model organisms, such as D. melanogaster and C. elegans mutations that influence yeast life span have been found to exert their effects through different physiological and genetic pathways, including those that participate in caloric restriction, gene silencing, genomic stability, growth regulation, mitochondrial function, and stress response (45-47,52,53). Replicative aging is undoubtedly a complex process, even in a eukaryote as simple as S. cerevisiae Different hypotheses have been proposed to explain yeast replicative aging. One hypothesis proposed by Sinclair and Guarente (54) posits that

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10 replicative aging is caused by progressive accumulation of extrachromosomal rDNA circles ( E R C s) in yeast mother cells. According to this model, E R C s are produced stochastically by intrachromosomal homologous recombination at the rDNA locus and are inherited asymmetrically by mother cells, which leads to E R C accumulation and replicative senescence. The rDNA locus in S. cerevisiae consists of a tandem array of ~150, 9.1 kb direct repeats, each of which encodes the four rRNAs (18S, 5.8S, 25S, and 5S) in precursor form. Many aspects of the E R C model have been supported experimentally. Numerous studies support the view that E R C s are produced by homologous recombination, are self-replicating, are inherited asymmetrically, and accumulate in mother cells (45,54,55). More controversial is the role E R C s play in the aging process. Are E R C s mediators or markers of yeast aging? Certain findings link E R C production with regulation of life span and support a mediator role for E R C s. One of the first life span extending mutations characterized in yeast ( SIR4-42 ) was found to redirect silent information regulator (Sir) protein complexes to the rDNA locus and limit recombination (51,56). Expression of SIR2 which encodes a nucleolar NAD-dependent histone deacetylase, correlates with longevity. Sir2p binds to rDNA and suppresses rDNA recombination and E R C production (57-59). Deletion of SIR2 shortens life span, whereas overexpression of SIR2 extends life span (60). FOB1 encodes a nucleolar fork blocking protein that binds to the replication fork barrier (RFB) site in rDNA and in so doing halts DNA replication in the direction opposite of pre-35S rRNA transcription (6163). The RFB site and the overlapping HOT1 site promote rDNA recombination (63,64). Mutations in (or deletion of) FOB1 reduce rDNA recombination, lower E R C levels, and

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11 extend life span (65). Recombination of replication forks stalled at RFB sites is suppressed by Sir2p (66) which partly explains the role of Sir2-dependent silencing in extending life span. Also, introduction of a plasmid carrying a stretch of rDNA, as an artificial E R C was shown to reduce life span (54). On the other hand, E R C s have been interpreted as a marker of aging that are a consequence, not a cause, of aging. Mutations that impair DNA replication, recombination, or repair have been observed to reduce life span without concomitant accumulation of E R C s (67-69). However, reduction in life span may be the result of the combined effects of age-dependent and age-independent processes at work in certain mutants. The hrm1 mutants, which affect rDNA recombination, age prematurely due to a combination of the normal aging process and a G 2 -like cell cycle arrest (69) Similarly, sgs1 mutants exhibit a shortened life span because of the combined effects of the normal aging process and cell cycle arrest due to defective recombination (70). Some petite mutants have been shown to have elevated E R C levels (71), but extended life spans (72). However, to our knowledge, both elevated E R C levels and extended life span in petite mutants have not been demonstrated side-by-side in the same strain. A sir2 mutant with an extended life span was reported to have normal E R C levels (73). More generally, the effects of SIR2 on life span have been attributed to altered patterns of gene expression, including altered transcription of rDNA, which may lead to an imbalance in ribosome synthesis (74,75). Thus, although there is agreement that the rDNA locus plays a key role in the yeast aging process, the precise role of extrachromosomal DNAs remains controversial.

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12 To shed light on this controversy, we have developed a plasmid-based model system to investigate the role of episomal DNAs in reduction of yeast life span. Here we present the first comprehensive test of the E R C model of yeast aging proposed by Sinclair and Guarente (54) We constructed three types of recombinant plasmid for this purpose: ARS plasmids, ARS/CEN plasmids, and 2 origin plasmids. ARS plasmids are most like E R C s in that they are circular DNA molecules with a replication origin but lack a cis -acting partitioning sequence. Classic pedigree analysis studies by Murray and Szostak showed that ARS plasmids exhibit a strong bias to be retained in mother cells during mitosis (43). Thus, ARS plasmids are predicted to accumulate in mother cells like E R C s, but this has not yet been demonstrated. ARS/CEN plasmids contain a centromeric DNA region that acts in cis to attach plasmid DNA to the mitotic spindle and ensure efficient delivery to daughter cells during mitosis. ARS/CEN plasmids should not accumulate in mother cells. 2 origin plasmids typically contain a DNA replication origin, a cis -acting REP3/STB element, and one copy of an inverted repeat that regulates plasmid copy number (~20 to 40 copies/cell) (76). The REP3/STB element actively partitions plasmid DNA to daughter cells during mitosis in cir + yeast strains (i.e., in strains that contain the endogenous 2 circle DNA plasmid that encodes proteins that interact in trans with REP3/STB ) (76). 2 origin plasmids are not predicted to accumulate in mother cells, although the 2 plasmid partitioning machinery is not predicted to exhibit the fidelity of a centromere-based partitioning machinery. We also constructed a series of plasmids containing functional rDNA repeat units, and tested their effects on life span. This represents a significant improvement over a previously reported

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13 experiment (54) which employed a non-functional stretch of rDNA (i.e., rDNA incapable of being transcribed to yield full-length 35S pre-rRNA). Roles of Different Cis-Acting Plasmid Sequences in Reduction of Yeast Replicative Life Span To study the effects of plasmids on yeast replicative life span, we generated two series of plasmids based on commonly-used integrating vectorspRS306 and pRS305 (77). In each plasmid, we inserted ARS1 or ARS1 and CEN4 or the 2 circle origin (see Materials and Methods). ARS1 (autonomous replicating sequence 1) is a nuclear genomic DNA replication origin whose function and domain organization have been studied in detail (78). Centromeric DNA from chromosome IV ( CEN4 ) has been mapped and functionally dissected (79). The region of the 2 circle plasmid extending from Table 2-1. Plasmids used in this study. Plasmid Origin, Insert Marker Backbone pJPA105 2 rDNA repeat (XmaI endpoints) TRP1 pAF15 pJPA106 2 rDNA repeat (AhdI endpoints) TRP1 pAF15 pJPA107 2 rDNA repeat (PsiI endpoints) TRP1 pAF15 pJPA113 ARS1 URA3 pRS306 (77) pJPA114 rDNA ARS URA3 pRS306 pJPA116 ARS1 CEN4 URA3 pRS306 pJPA117 rDNA ARS, CEN4 URA3 pRS306 pJPA138 2 URA3 pRS306 pJPA133 ARS1 LEU2 pRS305 (77) pJPA136 ARS1 CEN4 LEU2 pRS305 pJPA148 2 LEU2 pRS305 REP3 through the adjacent 599 bp 2 repeat functions as a replication origin as well as a cis -acting plasmid partitioning element (76,80). The plasmids used in this study are summarized in Table 2-1.

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14 To evaluate effects on life span, plasmids were transformed into strain W303AR5 (54). For each plasmid, six independently-isolated transformants were analyzed in parallel, and each life span curve reflects their collective behavior. Selection for the plasmid was maintained during life span analysis. Virgin mother cells unable to give rise to 5 daughters were discarded to exclude contributions from mother cells without plasmid. To identify mother cells that stopped dividing due to plasmid loss, rather than senescence, cells that had not divided in 2 days were transferred to nonselective medium and monitored for cell division and colony formation. A low percentage (<10%) of mother cells were found to give rise to colonies, and were excluded from the life span data set. Life span plates were incubated during the daytime at 30C, but placed overnight (~12 hours) at 14C, which gave a slightly, but significantly (p<0.01) longer life span than observed on plates stored overnight at 4C (Figure 2-1A). Interestingly, transformants harboring pJPA113 ( ARS1 ) showed dramatic reductions in both average and maximum life span compared to the Ura + control strain yAF6 (Figure 2-1B). yAF6 differs from pJPA113 transformants only in terms of plasmid DNA topology (i.e., integrated in yAF6 and episomal in transformants). Transformants containing pJPA116 ( ARS1, CEN4 ) have a reduced average life span compared to yAF6, but exhibit a maximum life span similar to yAF6 (Figure 2-1B). Thus, addition of a CEN4 element to an ARS1 plasmid suppresses reduction in maximum life span, but does not completely compensate for, or protect against, effects on average life span. Plasmids containing the 2 circle origin of replication were also constructed and analyzed. Yeast cells harboring pJPA138 (2 ori) show a reduction in both average and maximum life

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15 span (Figure 2-1B). Generally speaking, the extent of reduction in average and maximum life span in pJPA138 (2 ori) transformants is intermediate between that Figure 2-1. Life span analysis of plasmid-transformed yeast. Number of daughter cells (generations) produced per mother cell are plotted as a function of mother cell viability. A) Life span curves of strain W303AR5 (54) grown on SD (synthetic dextrose) and S+D (dextrose added after autoclaving) media at 30C during the daytime and stored overnight (~12 hours) at 4C or 14C. The number (n) of mother cells analyzed per curve is as follows: SD 4C, n=60; SD 14C, n=59; S+D 14C, n=60. B) Life span curves of W303AR5 transformed with plasmids pJPA113 ( ARS1 ), pJPA116 ( ARS1, CEN4 ), or pJPA138 (2 ori), and control strain yAF6 ( URA3 ) (n=55, 47, 57, and 58, respectively). C) Life span curves of W303AR5 transformed with plasmids pJPA133 ( ARS1 ), pJPA136 ( ARS1, CEN4 ), pJPA148 (2 ori), and control strain yAF5 ( LEU2 ) (n=38, 33, 41, and 59, respectively). D) Life span curves of W303AR5 transformed with pJPA116 ( ARS1 CEN4 ) determined on SD and YPD (n=45 and 49, respectively). Life spans of control strains yAF6 and W303AR5 were determined on YPD (n=50 and 55, respectively). Plasmids are described in Table 2-1. observed in pJPA113 ( ARS1 ) and pJPA116 ( ARS1, CEN4 ) transformants (Figure 2-1B). The results from multiple life span experiments are summarized in Table 2-2.

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16 Table 2-2. Life span data summary. Plasmid/Strain Mean Life Span Maximum Life Span n* pJPA113 12.4 1.8 21.8 2.2 4 pJPA116 23 1.4 39 2.7 4 pJPA138 16.3 1.8 31.3 0.6 3 yAF6 33.2 3.0 42 1 3 The results reported above were obtained with plasmids carrying a URA3 selectable marker. To eliminate the possibility that effects of plasmids on life span were due to URA3 or medium lacking uracil, we constructed plasmids with a LEU2 selectable marker (Table 2-1), and conducted life span experiments on medium lacking leucine. The results obtained with the LEU2 plasmid series were very similar to results obtained with the URA3 plasmid series (Figure 2-1C). pJPA133 ( ARS1 ) caused dramatic reductions in average (9.9 generations) and maximum (17 generations) life spans compared to the Leu + control strain yAF5. yAF5 yielded an average (30.3 generations) and a maximum (44 generations) life span very similar to the average and maximum lifespan for yAF6 (Table 2-2). Transformants containing pJPA136 ( ARS1, CEN4 ) yielded a maximum life span of 38 generations, but an average life span of 24 generations, similar to what was observed for the URA3 plasmid pJPA116 ( ARS1, CEN4 ). Plasmid pJPA148 (2 ori) reduced the average (15.5 generations) and maximum (31 generations) life span to an extent intermediate between pJPA133 ( ARS1 ) and pJPA136 ( ARS1, CEN4 ) (Figure 2-1C), similar to what was observed with the URA3 plasmid pJPA138 (2 ori) (Figure 2-1B). The reduction in average life span by ARS1 CEN4 plasmids pJPA116 and pJPA136 was unexpected. A similar plasmid had previously been reported to have no effect on life span when grown on YPD medium (54) One possible explanation for this

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17 difference was that ARS1 CEN4 plasmids are occasionally lost from mother cells, causing them to cease division on selective medium prior to senescence, which would result in a reduction in average life span. To test this, ARS1 CEN4 plasmid transformants were analyzed on non-selective YPD medium as done previously (54). On YPD, transformants carrying pJPA116 ( ARS1 CEN4 ) were as long-lived as control strains yAF6 ( URA3 ) and W303AR5 (Figure 2-1D). pJPA116 transformants analyzed in parallel on selective SD medium showed a reduction in average life span (Figure 2-1D), as expected. These findings support the interpretation that ARS1 CEN4 plasmids, which are present at near-unit copy number in transformants (see below), are occasionally lost from mother cells, rendering them unable to divide at a point in their life span prior to normal senescence. We have also examined the effects of two well-known plasmids that carry the TRP1 selectable marker. pTV3 carries the 2 origin whereas pRS314 carries ARSH4 and CEN6 (77,80). Life spans of transformants containing each plasmid were analyzed on medium lacking tryptophan. pTV3 transformants had an average life span of 18.7 and a maximum life span of 32, both values of which are in good agreement with corresponding values for the 2 origin plasmids pJPA138 and pJPA148 (Figure 2-1 and Table 2-2). pRS314 had average and maximum life spans of 21 and 41, respectively, which are in good agreement with values obtained with the ARS1/CEN4 plasmids pJPA116 and pJPA136 (Figure 2-1 and Table 2-2). These data allow us to exclude a specific role for ARS1 and CEN4 in life span reductions presented above (Figure 2-1). Plasmid Inheritance Correlates with Reduction in Yeast Life Span The plasmids used in this study were constructed to explore relationships between plasmid inheritance and effects on life span. Mitotic stability and plasmid copy number

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18 are widely-used measures of plasmid DNA inheritance. Mitotic stability is defined as the proportion of a population of cells grown under selection that contains plasmid. We determined the mitotic stability and plasmid copy number of the plasmids used in life span experiments. Included in our studies were plasmids containing the rDNA ARS. rDNA repeats contain a single, relatively weak ARS (81) pJPA114 and pJPA117 contain the rDNA ARS at the same position as ARS1 in pJPA113 and pJPA116, respectively (see Table 2-1 and Materials and Methods). Plasmid pJPA113 ( ARS1 ) was found to have a mitotic stability of approximately 20% (Figure 2-2A), which is typical of yeast replicating plasmids containing ARS1 which exhibit a mother cell partitioning bias (43) pJPA116 ( ARS1, CEN4 ) exhibited a much higher mitotic stability, ~90%, which is consistent with the presence of CEN4 centromeric DNA, and agrees with the mitotic stability of pRS316 ( ARSH4, CEN6 ) (Figure 2-2A). pJPA138 (2 ori) showed a high degree of mitotic stability, ~90% (Figure 2-2A). The 2 origin plasmid pRS424 had a somewhat lower mitotic stability by comparison (Figure 2-2A). pJPA114 (rDNA ARS) has a very low mitotic stability, <1% (Figure 2-2A). The presence of CEN4 with the rDNA ARS in pJPA117 improves mitotic stability to ~35% (Figure 2-2A). These results with pJPA114 and pJPA117 are consistent with the low efficiency of the rDNA ARS (81) Not surprisingly, it was impractical for us to carry out life span analyses of transformants containing pJPA114.

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19 Figure 2-2. Plasmid inheritance studies. Plasmids are denoted by cis -acting element(s). See Table 1 for plasmid descriptions. pRS316 ( ARSH4, CEN6 (77)) and pRS424 (2 ori, TRP1 (82)) are included for comparison purposes. A) Mitotic stability determinations. Mitotic stability is defined as the percentage of colony forming units in a culture grown under selective conditions that contains plasmid-borne selectable marker. Side-by-side bars are determinations from separate experiments. Average and standard deviation values are plotted. B) Plasmid copy number in toto for cell population. Average and standard deviation values from Southern blots of genomic DNA digested with BamHI (filled bars) and PstI (open bars) are shown. C) Plasmid copy number on a per cell basis. Values were calculated by dividing copy number values from panel B by mitotic stability values from panel A (average of both experiments). The variances in copy number values were determined, assuming a log normal distribution of values. Variances for all values were near 1.0, with the exception of rDNA ARS plasmid copy number, which had a variance of 3.0, which is indicative of a higher level of error in this measurement. Plasmid copy number was determined using Southern blot analysis. Copy number was displayed either as the total number of plasmids compared to the total number of genomes (copy number in the population, Figure 2-2B) or the total number of plasmid compared to the fraction cells (genomes) that contain a copy of the plasmid (copy number per cell, Figure 2-2C) by using a plasmids mitotic stability. Copy number determinations using two different restriction enzymes gave comparable results (Figure 2-2B). pJPA113 ( ARS1 ) exhibited the highest plasmid copy number (Figure 2-2C). Plasmids pJPA116 ( ARS1, CEN4 ) and pJPA117 (rDNA ARS, CEN4 ) exhibited near-unit copy number

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20 values (Figure 2-2C), which is typical of centromeric plasmids (79) such as pRS316 ( ARSH4, CEN6 ) (77) pJPA138 (2 ori) exhibited a copy number of ~33 (Figure 2-2C), which is in the range of copy number values reported for other 2 origin plasmid vectors (80). The high copy number of pJPA113 is primarily due to the asymmetric inheritance of this plasmid and its accumulation in mother cells, rather than ARS strength per se We reach this conclusion because pJPA114, which contains a weak (rDNA ARS) replication origin, achieves a copy number almost as great as pJPA113, which contains a strong ( ARS1 ) replication origin (Figure 2-2C). Thus, pJPA113 demonstrates a correlation between extent of reduction of transformant life span (Figure 2-1B) and tendency to be inherited asymmetrically and attain a high copy in yeast cells (Figure 2-2C). Plasmids Do Not Significantly Increase ERC Levels The results presented above suggest that reduction in life span by the ARS1 plasmid pJPA113 is due to asymmetric inheritance and accumulation in mother cells. An alternative explanation is that pJPA113 increases E R C levels in transformed cells, and thereby reduces life span indirectly. To address this possibility, we measured recombination at the rDNA locus using an ADE2 marker loss assay and measured E R C levels in transformed cells by Southern blotting. To analyze the frequency of recombination at the rDNA locus, we took advantage of the fact that W303AR5 contains ADE2 integrated at the rDNA locus (54). Recombination between flanking rDNA repeats results in loss of ADE2 and a change in colony color. The frequency of half-red sectored colonies is a measure of rDNA recombination rate (events per cell division). Transformation of yeast with plasmid results in a small increase in rDNA recombination as measured by ADE2 marker loss. For W303AR5, we find that ADE2 marker loss occurs at a frequency of ~1.3 per

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21 thousand cell doublings (Figure 2--3A), which is in good agreement with frequencies reported by others (60,68,69) The rate of ADE2 marker loss from yAF6 ( URA3 ) occurs at ~2.7 per thousand (Figure 2-3A). Transformants containing the three plasmids used in this study, pJPA113 ( ARS1 ), pJPA116 ( ARS1, CEN4 ), and pJPA138 (2 ori), exhibited marker loss rates of 4.1, 4.5, and 4.1 per thousand cell doublings, respectively. The differences between transformants and yAF6 represent increases of less than 2-fold. Higher levels of ADE2 marker loss are typically observed in strains with reduced life spans. For example, short-lived sir2 mutants exhibit ADE2 marker loss rates >10-fold higher than isogenic SIR2 strains (60). To directly compare E R C levels, yeast transformants and control strains were analyzed by Southern blotting, and E R C monomer bands were quantitated (see Materials and Methods). E R C monomers consist of a single 9.1 kb rDNA repeat and were chosen for purposes of quantitation because they are well-resolved from chromosomal rDNA and other E R C bands on Southern blots. E R C monomer levels in transformants were not significantly different than E R C monomer levels in control strains. Control strains W303AR5 and yAF6 ( URA3 ) have approximately 0.0007 and 0.0015 E R C monomers per total chromosomal rDNA, respectively (Figure 2-3B). Transformants bearing pJPA113 ( ARS1 ), pJPA116 ( ARS1, CEN4 ), and pJPA138 (2 ori) have E R C monomers levels of 0.0014, 0.001, and 0.001, respectively (Figure 2-3B). These values are within the error of measurements and are not significantly different (Figure 2-3). For comparison, we examined yAF5 ( LEU2 ), which contains a copy of pRS305 integrated at the leu2-113 locus, and found that the E R C monomer level was 0.001, which is intermediate between W303AR5 and yAF6 (Figure 2-3B). Quantitation of slower

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22 migrating E R C multimer bands did not reveal significant differences in levels between transformant and control strains (data not shown). We conclude that plasmids do not have a significant effect on E R C levels. Figure 2-3. Extrachromosomal rDNA circle ( E R C ) formation in yeast transformants. Plasmids are denoted by cis -acting element(s). See Table 2-1 for plasmid descriptions. Control strains W303AR5 ( W303 ), yAF5 ( LEU2 ), and yAF6 ( URA3 ) did not contain plasmid. A) ADE2 marker loss assay. The number of half-red sectored colonies on minimal selective medium per total colony number defines the per (first) cell division rate of loss of the ADE2 marker from the rDNA repeat in W303AR5. Total number (n) of colonies scored is shown. B) E R C monomer levels. Southern blotting analyses of DNA from transformed and control strains grown on selective media were done to quantify chromosomal rDNA and E R C monomer band levels (see Materials and Methods). E R C monomer band intensity was divided by chromosomal rDNA band intensity to give a normalized E R C monomer/chromosomal rDNA ratio. Average and standard deviation values are plotted. Plasmid Accumulation Correlates with Reduction in Life Span If plasmids reduce life span in a manner analogous to E R C s, then plasmid DNAs should accumulate in old mother cells. To test this prediction, we used a biotinylation and magnetic sorting approach to isolate ~7-generation old yeast cells (see Materials and Methods). Plasmid DNA levels in young and old cells were measured by quantitative Southern blotting. The ages of old and young (unsorted) cells were determined by counting bud scars stained with Calcofluor (83) From single sort experiments, the average ages of

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23 yeast transformed with pJPA113, pJPA116, pJPA138, and yAF6 were 6.9, 7.0, 6.1, and 6.2 generations, respectively (Figure 2-4A). Young cells from the same cultures were an average of 1.5, 1.4, 1.1, and 1.1 generations old, respectively (Figure 2-4A). Inspection of the Southern blot clearly reveals increases in relative amounts of pJPA113 ( ARS1 ) and pJPA138 (2 ori) in old cells (Figure 2-4B). pJPA116 ( ARS1, CEN4 ) did not accumulate in old cells, and yields bands similar in their intensities to corresponding bands from yAF6 (Figure 2-4B). In a striking illustration of the accumulation of pJPA113 and pJPA138 in old cells, the linearized plasmid DNA bands can be observed by ethidium bromide staining (Figure 2-4D). E R C levels in young and old cells were also analyzed by Southern blotting. Hybridization to rDNA probe revealed E R C bands and a broad band corresponding to the rDNA locus on chromosome XII (Figure 2-4C). We note that all old cell preparations contained increased numbers of both monomeric and slower-migrating E R C species (Figure 2-4C). The E R C and rDNA repeat bands collapse to a single 9.1 kb band following digestion with KpnI, which cuts rDNA once (data not shown). Chromosomal and plasmid band intensities were quantitated using a PhosphorImager. Consistent with our determinations in Figure 2-2, pJPA113 ( ARS1 ) and pJPA138 (2 ori) are present at high copy number in young cells, but pJPA116 ( ARS1, CEN4 ) is not (Figure 2-4E). In ~7-generation old transformants, the plasmid copy numbers for pJPA113 and pJPA138 are dramatically increased, reaching values of 254 and 137, respectively (Figure 2-4F). This represents a difference in copy number between young and old cells of ~13-fold for pJPA113 and ~6-fold for pJPA138. By

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24 Figure 2-4. Plasmid DNA and extrachromosomal rDNA circle ( E R C ) levels in young and old cells. Panel A conveys the cis -acting elements present in each plasmid (see also Table 2-1). Plasmids are abbreviated by numbers in panels B-H. All plasmids carry URA3 Control strain yAF6 ( URA3 ) did not contain plasmid. Old cells were harvested using a biotinylation and magnetic sorting approach (see Materials and Methods). A) Age profile histograms of young and old cells. Number of cells is plotted as a function of number of bud scars (n>40 for each histogram). B) Southern blot of plasmid DNAs. PstI-digested genomic DNA yields a 3.67 kb URA3 band. Other bands are plasmid-derived. Genomic URA3 DNA in lane Old 113 migrated as two bands due to partial over-digestion of this sample. C) Southern blot of E R C s. D) Ethidium

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25 bromide stained agarose gel corresponding to the blot in panels B and C. DNA marker sizes (in kb) are shown. E and F. Plasmid levels in young and old cells (quantitation of data presented in panel B). G and H) E R C monomer levels in young and old cells (quantitation of data presented in panel C). For E-H, ratios of episome (plasmid or E R C monomer) band intensity divided by chromosomal rDNA band intensity (X1000) are plotted (on a semi log scale). See Fig. 2-1B for corresponding life span data. Comparable results were obtained from similar cell sorting and Southern blotting experiments and are discussed in the Results section. comparison, 7-generation old pJPA116 transformants show no significant increase in plasmid copy number (Figure 2-4F). In a separate experiment with pJPA113 and pJPA116 transformants, in which genomic DNA was digested with BamHI instead of PstI, quantitative analysis revealed that young cells contained 27 and 1.5 plasmids/cell, respectively, whereas old cells contained 283 and 1.2 plasmids/cell, respectively (data not shown). This corresponds to a ~10-fold increase in plasmid copy number for pJPA113 in ~7-generation old cells, and no significant increase in pJPA116 copy number, which agrees with findings presented in Figure 2-4E, F. E R C monomer levels were also quantitated in young and ~7-generation old transformants and yAF6. E R C monomer levels in young cells were equal or close to 0.001 (Figure 2-4G), which agrees with measurements presented above (Figure 2-3B). In old cells, however, E R C monomer levels were appreciably higher, and exhibited increases between ~20-fold to ~70-fold (Figure 2-4H). The levels of E R C s we observe in ~7-generation old cells appears comparable to E R C levels in sorted cells of similar age reported by others (e.g., (54,60)), although quantitative analysis of E R C levels in young and old yeast cells is not commonly reported in the literature. In the experiment shown in Figure 2-4, E R C monomer levels in yAF6 ( URA3 ) are higher than in transformants (Figure 2-4H). This raises the question: does the presence

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26 of plasmid reduce E R C levels? In a separate experiment, E R C monomer levels in young cells were equal or close to 0.001 ( E R C monomer/chromosomal rDNA) and E R C monomer levels in old transformants containing pJPA113, pJPA116, and pJPA138 and in old yAF6 cells were determined to be 0.083, 0.075, 0.045, and 0.081, respectively (data not shown). The similar E R C levels in yAF6 and transformants in this experiment suggest that plasmid vectors do not appreciably affect E R C monomer levels (Figure 2-5). Does the extent of E R C accumulation in old cells in Fig. 4 agree with predictions based on our estimates of rates of recombination within the rDNA locus (see above, Figure 2-3)? If we assume that extrachromosomal rDNA repeats are generated at a rate of 0.5 per cell per generation, and that E R C s are retained in mother cells, then 6-7 generations should yield an increase of between 32to 64-fold, which is similar to the observed range of increase from 20to 70-fold (Figure 2-4G, H). We have also quantitated the relative amount of all E R C s (i.e., monomers, multimers, and concatemers) found in old transformants containing pJPA113, pJPA116, and pJPA138 and in old yAF6 cells. We found levels, respectively, of 0.140, 0.136, 0.086, and 0.238 (extrachromosomal rDNA/chromosomal rDNA; data not shown). These values mirror levels of accumulation of E R C monomers presented in Figure 2-4H. Thus, E R C monomers comprise approximately 1/4 to 1/3 of all extrachromosomal rDNA repeats and are present at similar levels relative to all E R C s in old transformed and untransformed cells. To extend these studies, yeast sorting experiments were done with transformants containing the LEU2 plasmids pJPA133 ( ARS1 ), pJP136 ( ARS1, CEN4 ), and pJPA148 (2 ori), and with the LEU2 strain yAF5. The average ages of sorted yeast transformed

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27 with pJPA133, pJPA136, pJPA148, and yAF5 are 7.1, 7.0, 7.6, and 7.9 generations, respectively (Figure 2-5A). Young cells from the same cultures were an average of 1.7, 1.0, 1.6, and 1.6 generations, respectively (Figure 2-5A). pJPA133 and pJPA148 attain copy number levels of 119 and 39, respectively, in ~7-generation old cells (Figure 2-5C). This represents an increase in copy number between young and old cells of ~30and ~8fold for pJPA133 and pJPA148, respectively. pJPA136 did not show a significant increase in old cells (Figure 2-5C). In comparison to pJPA113 and pJPA138, pJPA133 and pJPA148 reached lower absolute levels of plasmid in ~7-generation old cells. However, pJPA133 and pJPA148 accumulated to similar extents in terms of foldincrease. To resolve if this difference in absolute levels of plasmids in old cells was due to experimental error, sorting experiments with transformants and the control strain were repeated, followed by Southern analyses. The repeat experiment gave results very similar to first experiment, both in terms of absolute level of plasmid in young and old cells as well as fold-increase in young and old cells (Figure 2-5B, C). This indicates that plasmids with identical ARS1 origins and CEN4 elements, but with different backbones and selectable markers, are maintained at different absolute copy number levels in young and old cells. Nevertheless, similar fold-differences in plasmid levels are observed between young and ~7-generation old cells. This indicates that ASR1 and CEN4 elements present on plasmids functionally determine patterns of plasmid inheritance and accumulation during yeast mother cell replication. Next, E R C monomer levels in transformants containing pJPA133, pJPA136, and pJPA148, and in strain yAF5 were quantitated. In young cells, E R C monomers were detected at relatively high levels (Figure 2-5C). However, E R C monomer levels in ~7

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28 generation old cells were similar to levels observed above for pJPA113, pJPA116, and pJPA138 transformants (compare Figures 2-4H and 2-5D). Thus, E R C s in the Leu + transformants showed accumulation over a range of ~3-fold to ~12 fold between young and old transformants. This range of fold-increase is approximately 7-fold lower than the ~20-fold to ~70-fold increase in E R C levels between young and old Ura + transformants. This suggests that the rate of E R C accumulation during the aging process is regulated so that old cells of similar ages contain similar levels of E R C s despite differences in initial levels of E R C s in young cells. Figure 2-5. Plasmid DNA and extrachromosomal rDNA circle ( E R C ) levels in young and old cells. Plasmids are abbreviated by numbers in panels B-E. Panel A conveys the cis -acting elements present in each plasmid (see also Table 2-1). All plasmids carry LEU2 Control strain yAF5 ( LEU2 ) did not contain plasmid. Data were collected as described in Fig. 4. A. Age profile histograms of young and old cells. B and C. Plasmid levels in young and old cells (semi log plot). Data from two Southern blotting experiments are shown (Exp 1 and Exp 2). D and E. E R C monomer levels in young and old cells (semi log plot). See Fig. 1C for corresponding life span data.

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29 An important trend emerges from our studies of plasmid accumulation in old cells. Plasmids that accumulate to the greatest degree in old cells (Figures 2-4 and 2-5) exert the most profound effect on life span (Figure 2-1). ARS1 plasmids attain the highest copy numbers in old cells and have the most pronounced effect on life span. ARS1/CEN4 plasmids maintain a copy number near unity in young and old cells and have a small effect on maximum lifespan and a moderate effect on average life span. Plasmids with 2 origins attain a copy number in old cells roughly half that of ARS1 plasmids and reduce life span roughly half as much as ARS1 plasmids. This suggests the existence of an inverse relationship between plasmid accumulation in old cells and reduction in yeast life span. Terminal Cell Morphology Currently, in the field of yeast aging, there are few approaches available to directly address the senescent phenotype in old non-dividing cells. To address this issue indirectly, we scrutinized the terminal morphology of cells at the end of their life span (Appendix A-H). The rationale for this approach is that cell morphology is a phenotypic indicator of cell cycle stage and can serve as a basis to compare senescent cells (70) If cell morphology in terminal transformed cells is very different from the morphology of terminal wild type cells, this would imply that different mechanisms may bring about the senescent phenotype in transformed and untransformed cells. To examine terminal yeast cells, images of terminal cells were collected from three different life span experiments. Three different cell morphologies were scored: unbudded cells, single-budded cells with small buds, and single-budding cells with large buds (70). Bud emergence in S. cerevisiae correlates with entrance into S phase, and

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30 small buds are indicative of early S phase, whereas large buds are indicative of late S/G 2 or mitotic arrest. Unbudded cells are in G 1 phase. Between 10-15% of the terminal cells, transformed or untransformed, had multiple buds (data not shown) and were omitted from this comparison. For pJPA113 ( ARS1 ) and pJPA116 ( ARS1, CEN4 ) transformants, and W303AR5, more than 50% of terminal cells were unbudded (Figure 2-6). Typically, Figure 2-6. Terminal morphology of senescent cells. Cells at the end of life span experiments were classified according to budding pattern as described (70). Small buds were defined as having a diameter less than 25% of the diameter of the mother cell. All other buds were classified as large. Average and standard deviation values from three independent experiments are shown (n >40 for each transformant or control strain in each experiment). between 50% and 60% of senescent yeast cells have been found to be unbudded (69,70). pJPA116 transformant cells consistently yielded the highest proportion (~65%) of unbudded cells (Fig. 6). yAF6 ( URA3 ) and pJPA138 (2 ori) transformants ceased dividing with a predominance, yet a lower percentage, of unbudded cells (Figure 2-6).

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31 Thus, the majority of pJPA113 transformants, like W303AR5 cells, senesced in G 1 as expected. In addition, similar proportions of small budded and large budded terminal cells in senescent pJPA113 transformants and W303AR5 cells (Figure 2-6) indicate that similar proportions of these cells arrested in similar phases (S or G 2 /M) of the cell cycle. Thus, this analysis supports the interpretation that pJPA113 ( ARS1 ) reduces life span by a normal aging process. Do Functional rDNA Transcriptional Units Play a Role in Reduction in Life Span? Although plasmids without rDNA sequences reduce yeast life span, it is important to consider a potential role for rDNA sequences in life span reduction. It is possible that E R C s reduce life span in a manner that is mechanistically more complex than the manner in which plasmid episomes reduce life span. There are significant differences in coding potential between plasmids and E R C s. The 9.1 kb rDNA repeat carries genes for rRNA precursors as well as the gene TAR1 which lies on the strand opposite the 25S rRNA and encodes a mitochondrial protein (84). One way to address this issue is to ask whether or not a plasmid vector carrying an rDNA repeat unit has a more pronounced effect on life span than plasmid vector alone. It is important to note this issue was not completely addressed in a previous study employing the rDNA-containing plasmid pDS163 (54) Plasmid pDS163 does not contain a functional 9.1 kb rDNA repeat unit. The rDNA on pDS163 consists of a 12.1 kb insert extending from an EcoRI site within the coding sequence of 5.8S rRNA to the 5-most EcoRI site in the 25S rRNA coding region (data not shown). The 12.1 kb fragment does not carry a full-length 35S prerRNA transcription unit and is capable of producing only a truncated 35S pre-rRNA transcript, which if processed would be incapable of yielding mature 25S rRNA.

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32 To determine if an episomal rDNA repeat influences life span, we constructed three plasmids containing 9.1 kb rDNA repeats and used them in life span experiments. The three plasmids, pJPA105, pJPA106, and pJPA107 contain 9.1 kb repeats with different endpoints in the plasmid pAF15, which contains a 2 origin (see Materials and Methods, and Table 2-1). Plasmid pJPA105 contains a repeat with XmaI end points, Figure 2-7. Life span analysis of yeast transformed with plasmids containing rDNA repeats. Number of daughter cells (generations) produced per mother cell are plotted as a function of mother cell viability. Life span analysis was done as described in Figure 2-1 using W303AR5 carrying plasmids pJPA105 (n=45), pJPA106 (n=43), or pJPA107 (n=46) and control plasmid pAF15 (n=46). Plasmids pJPA105, pJPA106, and pJPA107 contain full length (9.1 kb), rDNA repeats with different endpoints (see Table 2-1 and Materials and Methods). pJPA105 contains an rDNA insert with XmaI endpoints, which has been shown to be functional in vivo (85). which has been shown by Nomura and colleagues to functionally complement an rDNA deletion in vivo (85). pJPA106 and pJPA107 contain repeats with AhdI and PsiI endpoints, respectively, which should not interfere with rDNA gene expression (Figure 12). A 2 origin plasmid was used because plasmids constructed with rDNA inserts

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33 whose replication relied solely on the rDNA ARS were found to integrate into the chromosomal rDNA locus with high frequency (as determined by Southern blot analysis; data not shown). Life span determinations of W303AR5 transformants containing pAF15, pJPA105, pJPA106, and pJPA107 were done as described above (see Figure 21). pJPA105, pJPA106, pJPA107, and pAF15 transformants gave very similar life span curves, indicating that the presence of a functional rDNA repeat does not have a dramatic effect on life span (Figure 2-7). All four plasmids affect life span to an extent similar to the 2 origin plasmids pJPA138 and pJPA148 (Figure 2-1B, C), although the average life spans for pJPA105, pJPA106, pJPA107, and pAF15 (13.3, 11.8, 11.7, and 12.2 generations, respectively) are lower than the average life spans for pJPA138 and pJPA148 transformants (15.5 and 16.3 generations, respectively; Figure 2-1 and Table 22). Life span curves for pJPA106 and pJPA107 transformants did not show a statistically significant difference from pAF15 transformants based on the Wilcoxon signed pair rank test (p>0.05) Only transformants carrying pJPA105 and pAF15 exhibited a statistically significant difference (p<0.05), but this represents a small increase in life span of transformants carrying pJPA105. These findings support the conclusion that the presence of a full-length rDNA repeat per se does is not required for, and does not necessarily augment, reduction in yeast life span. Summary Our studies show that yeast plasmids accumulate in mother cells and reduce replicative life span. The effect of plasmids on life span appears to be a direct effect, and not an indirect effect on E R C levels in mother cells. A functional rDNA repeat unit is not required for reduction in life span, and the presence of a functional rDNA repeat does not augment reduction in life span by plasmids. Thus, plasmids containing ARS

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34 elements appear to mimic E R C -mediated reduction in life span. These findings provide strong evidence that replicative aging in S. cerevisiae is caused by accumulation of episomal DNA. The fact that functional rDNA sequences are not required for reduction in life span argues that expression of rDNA genes present on E R C s is not a causative process in yeast aging. This indicates that accumulation of episomal DNAs, such as ARS plasmids and E R C s, is one mechanism by which yeast life span is regulated.

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35 CHAPTER 3 TWO MICRON CIRCLE: A NATURALLY OCCURRING EPISOMES ROLE IN AGING One of the processes that has been proposed to regulate replicative life span in the budding yeast Saccharomyces cerevisiae is the accumulation of extrachromosomal rDNA circles (ERCs) by yeast mother cells (54) ERCs are generated by recombination within the rDNA repeat region on chromosome XII and are passed on to daughter cells infrequently due to an inheritance bias exhibited by replication origin-containing DNA episomes (43). We have shown that plasmids containing an autonomously replicating sequence (ARS; yeast DNA replication origin) reduce life span due to their accumulation during replicative aging (86). This suggests that DNA episomes in general regulate replicative aging, and reduce life span due to their accumulation in yeast mother cells. The majority of laboratory strains of S. cerevisiae contain an endogenous plasmid known as the two micron (2 ) circle, due to the length of its circular DNA determined by electron microscopy (76,87). Strains harboring this non-Mendelian genetic element are denoted cir + ; strains lacking it are referred to as cir 0 (88). Four genes and multiple cisacting sequences on the 2 micron plasmid have been mapped and functionally dissected (76,87). These are responsible for maintaining copy number at approximately 20-40 copies per cell by a recombination-based mechanism and ensuring high fidelity transmission of the 2 micron plasmid during cell division and mating (76,87,89). There are no significant growth phenotypes generally associated with the presence of the 2 micron plasmid in cir + strains, and conversely, negligible growth advantages conferred to

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36 cir 0 strains (76,87) This has led to the view that the 2 micron plasmid is a parasitic DNA that imposes only a minor selective disadvantage to host strains (76,87). However, previous studies have not examined the possibility of an effect of the 2 micron plasmid on replicative life span. A minor effect on replicative life span is not predicted to result in a discernable difference in vegetative growth rate, and may have been overlooked in the past. To address this issue we have taken advantage of a novel and simple method for curing a cir + yeast strain of 2 micron plasmids. Previously described methods (90,91) for curing strains of 2 micron plasmids are more time-consuming and are less convenient than the method described herein. New Method for Removal of Two Micron Circle During the course of plasmid copy number studies published elsewhere (86) we fortuitously observed that transformants containing recombinant yeast shuttle vectors with an rDNA ARS lost 2 micron plasmid DNA more frequently than was expected, based on the known inheritance behavior of 2 micron plasmids. In our initial Southern blotting studies, half of the transformants containing plasmid pJPA114 (4/8 transformants) or plasmid pJPA118 (2/4 transformants) lost 2 micron plasmid DNA (data not shown). Plasmids pJPA114 and pJPA118 are derived from pRS306 (77) and have been described previously (86). pJPA114 contains a 200 bp insert with the rDNA ARS; pJPA118 contains a complete 9.1 kb rDNA repeat with its ARS. The rDNA ARS has relatively weak replication origin activity due to the presence of a non-consensus ARS consensus sequence (ACS) (81). As a result, the majority of pJPA114 and pJPA118 transformants form colonies slowly on selective SD medium. In instances where fastgrowing pJPA118 colonies arose on transformation plates or streaks of individual transformants, Southern analysis revealed that pJPA118 had integrated into the rDNA

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37 Figure 3-1. Southern blot analysis of pJPA114 transformants. Panel A. DNAs from twelve transformants were digested with PstI, separated on a 1% agarose gel and stained with ethidium bromide. Size markers (M) are shown (in kb). Panel B. DNAs from the gel in panel A were transferred to a nylon membrane, hybridized to 32 P-labeled 2 micron plasmid DNA probe, and visualized with a PhosphorImager. The 6318 bp 2 micron plasmid contains one PstI site and yields a single 6.3 kb band (arrowhead). The faint bands above and below the 2 micron plasmid band likely correspond to hybridization to regions of homology in yeast chromosomes (e.g., a region of homology in Ch III yields a PstI fragment of 7747 bp, which corresponds to the size of the upper faint band). Longer exposures of the Southern blot revealed no detectable bands in lanes 8 and 9 (data not shown). locus (data not shown). Transformants containing pJPA118 were not studied further because of the relative frequency with which pJPA118 integrated into the rDNA repeat locus. Fast-growing pJPA114 transformants arose only very infrequently and were not analyzed for plasmid integration by Southern blotting. Transformants containing plasmid pJPA113 (86), which is derived from pRS306 but contains the ARS1 origin instead of the rDNA ARS, did not show loss of 2 micron plasmids in our Southern blotting studies (data not shown). ARS1 contains an ARS consensus sequence (ACS) that conforms to the

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38 consensus observed in most ARS elements and is considered a strong ARS, unlike the rDNA ARS. These preliminary results suggested that pJPA114 may be generally useful to cure cir + strains of 2 micron plasmid DNA. To further test the use of pJPA114 for this purpose, we transformed W303AR5 with pJPA114, streaked independently-isolated transformants to single colonies, obtained isolates lacking pJPA114, and analyzed twelve arbitrarily-chosen isolates by Southern blot analysis (see Materials and Methods). pJPA114 has two technical merits in this experiment. Because pJPA114 contains the rDNA ARS, it is readily lost from transformants grown in the presence of uracil. Loss of pJPA114 can be confirmed by growth on medium containing 5-fluoroorotic acid (5FOA). In this experiment, 2 of 12 yeast isolates lost 2 micron plasmid DNA (Figure 3-1). This confirms our initial findings that pJPA114 transformants lose 2 micron plasmids with a sufficiently high frequency to allow pJPA114 to be useful for curing a strain of the 2 micron plasmid. Two Micron Circle Does Not Reduce Life Span To determine if the presence of 2 micron plasmids influenced replicative life span, microdissection-based life span determinations were done as described (86,92). The two cir 0 strains, yAF7 and yAF8, obtained from the experiment presented in Figure 1 were compared to the parental strain W303AR5. No apparent difference in replicative life spans was observed (Figure 3-2). The average replicative life spans for yAF7, yAF8, and W303AR5 were 22.7 (.2), 21.9 (.3), and 23.5 (.9) generations, respectively. Wilcoxon two-sample paired signed rank tests revealed no statistically significant differences between the three life span curves (p<0.05). Thus, the presence of 2 micron

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39 plasmids in W303AR5 does reduce replicative life span compared to two independentlyisolated, otherwise isogenic strains lacking 2 micron plasmids. Figure 3-2. Life span analysis of cir + and cir 0 yeast. Number of daughter cells (generations) produced per mother cell are plotted as a function of mother cell viability. yAF7 and yAF8 are derivatives of W303AR5 (54) that lack the 2 micron plasmid, and correspond, respectively, to lanes 7 and 8 in Figure 3-1. The number of mother cells (n) analyzed for each curve equals 55, 56, and 55 for yAF7, yAF8, and W303AR5, respectively. The three curves are indistinguishable by Wilcoxon two-sample paired signed rank tests (p<0.05). Two Micron Circle Does Not Accumulate in Old Cells These findings indicate that 2 micron plasmids do not confer a disadvantage insofar as replicative life span is concerned. This suggests that 2 micron plasmids do not accumulate during the aging process. To test this prediction, ~6-generation old yeast cells were prepared by magnetic sorting (see Materials and Methods) and 2 micron plasmid DNA levels were analyzed by Southern blotting. No differences in 2 micron

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40 Figure 3-3. Two micron plasmid levels in young and old cells. Old cells were isolated by biotinylation and magnetic sorting (see Materials and Methods). Bud scars in young and old cells were stained with Calcofluor and counted to determine average age. DNAs from young and old cells were analyzed by Southern blotting as described for Figure 3-1. Size markers (M) are shown (in kb). To normalize levels of 2 micron plasmid DNA to genomic DNA levels, the blot was stripped and rehybridized to 32 P-labeled probe to URA3 2 micron plasmid and URA3 band intensities were quantitated, and no significant difference was found between the ratios of 2 micron plasmid DNA to URA3 DNA in young and old cells. plasmid DNA levels were observed between populations of cells with average ages of 1.1 and 6.2 generations (Figure 3-2). To normalize the amounts of 2 micron DNA present in young and old cell samples to the amounts of genomic DNA present, the relative amount of the URA3 gene was determined by Southern blotting (Figure 3-2). Quantitative analysis of the intensities of bands corresponding to 2 micron plasmid DNA and URA3 was done and revealed no significant difference between normalized 2 micron DNA

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41 levels in young and old cells (data not shown). Previous studies have shown substantial accumulation of ERCs (5to 50-fold) and non-centromeric recombinant plasmids (5to 25-fold) in 7-generation old yeast cells (86). Thus, 2 micron plasmids are unlike ERCs and non-centromeric yeast plasmid vectors, and do not accumulate in old cells. Summary In chapter 2, 2 micron origin plasmids were shown to accumulate. The same is not true for naturally occurring 2 micron circles. They do not accumulate; therefore, they do not reduce lifespan. While only confirming the model of plasmid aging, the loss of 2 micron circles in transformants of pJPA114 is very interesting. What is pJPA114 doing to cause 2 micron circle loss?

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42 CHAPTER 4 A CELLS LIMITED RESOURCES AND PLASMID COMPETITION The main hypothesis being developed to explain ERC mediated aging revolves around the idea that episomes cause a replication burden in cells. As cells age, they accumulate episomes (93). The episomes eventually reach a high copy number. It is so high, that it is about the same amount of DNA as the yeast genome (93). This means that an old cell is replicating two or more times the amount of DNA it normally replicates. This enormous amount of DNA could require all of a cells replication factors and DNA substrates to complete replication. If one of these factors or substrates is limiting, then the cell may encounter problems during replication. This could result in mutations, double strand breaks, etc. In order to further explore this idea, the following experiments were completed. Sml1p, Mec1p, RNR, and Rad53p Pathway Sml1p and Mec1p are involved in a well know DNA damage and repair pathway (94). Most importantly it senses replication fork slowing and stalls. The cascade starts by Mec1p sensing DNA damage or replication fork stalling. It then signals through Rad53p to Sml1p. Sm11p, an inhibitor, releases and thereby activates Ribonucleotide Reductase (RNR) (Figure 4-1). RNR makes dNTPs from NTPs by removing the 2 hydroxyl from ribose. This reaction results in an increase of cellular pools of dNTPs and the progression of stalled replication forks (94).

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43 Figure 4-1. Mec1p, Rad53p, Sml1p, and RNR pathway (94). This pathway ultimately leads to the production of dNTPs. SML1 Deletions Do Not Increase Life Span. To see whether an increase in the cellular dNTPs pools would counteract the affects of an episomes replication burden, a SML1 deletion was created with the insertion of HIS3 By removing the RNR complex inhibitor, Sml1p, dNTPs are continuously being

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44 produced no matter what the state of the cell. SML1 deletions are known to increase dNTPs levels in the cell by 2-3 fold. In addition to looking at the life span of SML1 and sml1 strains, transformants of sml1 were also test for an extended life span. The plasmid used was pJPA113. This is the ARS1 plasmid from Figure 2-1B and Table 2-1. Transformed cells may help to amplify the affect sml1 has on a replication burden, since they contain more episomes and have shorter life spans. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 10 20 30 40 Generations Fraction of Cells Viable yAF10+ARS yAF10 sml1 +ARS sml1 Figure 4-2. Life span of SML1 deletions. yAF10 ( HIS3 ; control) had a mean life span of 25.5 and a n=55. sml1 had a mean life span of 25.9 and a n=57. yAF10 + ARS (control) had a mean life span of 12.9 and a n=42. sml1 + ARS had a mean life span of 11.8 and a n=54. The ARS plasmid used was pJPA113 (ARS1). The SMLl1 deletion had the same life span as the control (yAF10). They were statistically indistinguishable using the Wilcoxon two-sample paired signed rank test.

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45 yAF10 is the yAF5 strain with its his3 locus repaired to control for insertion of HIS3 into sml1 The transformed sml1 cells also had the same life span as the transformed control strain (Figure 4-2). Reproducing previous results, the pJPA113 (ARS1) transformants had a mean life span within the standard deviations of the life spans in Table 2-2. Since replication is a doubling process, it is conceivable that an increase in dNTPs of 2-3 fold is not enough to be seen on a life span. In other words, the life span assay may not be sensitive enough. In old cells, episomes account for a large proportion of the total DNA (could be more than half). Since they are retained due to mother cell bias, a division occurring at an old age would increase the total DNA content by nearly double. The only DNA being passed on to the daughter cell would be the chromosomes, not the massively accumulated and newly replicated episomes. The key to this idea is that episomes can reach a quantity larger than that of the genome. A sml1 may only have increased a cells life span by one doubling, not enough to be observed by life span. Old Cells Do Not have an Increased Sensitivity to Hydroxyurea Hydroxyurea (HU) is a chemical inhibitor of the RNR complex (94) It has been widely used when studying Mec1p and Sml1p. Sm11 has an increased resistance to HU. Strains that are sm11 mec1 or sm11 rad53 have an increased sensitivity to HU. Interestingly MEC1 and RAD53 cannot be deleted without a SML1 deletion suppressing their lethality (94). Sml1p has a critical role in HU sensitivity because it is the inhibitor of the RNR complex. Without SML1 it takes more HU to suppress the larger pool of active RNR in the cell. If MEC1 or RAD53 are deleted then Sml1p does not release the RNR complex and the cell cannot make dNTPs. This is why sml1 is required when deleting MEC1 and RAD53 Mec1 sml and rad53 sml1 strains are not less sensitive to HU because of Mec1p and Rad53p regulation of transcription factors

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46 for the RNR proteins through Dun1p (Figure4-1). Without the activation of Dun1p, RNR levels in the cell stay the same and cannot compensate for the inhibition by HU. Figure 4-3. Hydroxyurea sensitivity of young and old cells from 0 mM to 200 mM. Each plate has five rows of pin stamps. Each row is a serial dilution of a strain. The first row is a WT control yAF6. The second row is a WT control W15884C. The third row is sml1 The fourth row is mec1 sml1 The fifth row is rad53 sml1 A change in HU sensitivity by old cells would show that this DNA repair pathway plays a role in the aging process. Two WT, sml1 mec1 sml1 and rad53 sml1 strains were magnetically sorted to get young and old cells. Serial dilutions were pin stamped onto minimal medium plates containing various levels of hydroxyurea. The first pin stamping contained HU concentrations of 0, 50, 100, 200, 300, and 400 mM. As expected the mec1 sml1 and rad53 sml1 strains had an increased sensitivity to HU, while the WT and sml1 were more resistant. There were no noticeable differences in the sensitivity of HU between the young and old cells (Figure 4-3). The 300 and 400 mM HU concentrations are not shown because no strains were able to grow in those conditions.

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47 Figure 4-4. Hydroxyurea sensitivity of young and old cells from 0 mM to 50 mM. Each plate has five rows of pin stamps. Each row is a serial dilution of a strain. The first row is a WT control yAF6. The second row is a WT control W15884C. The third row is sml1 The fourth row is mec1 sml1 The fifth row is rad53 sml1

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48 To further investigate HU sensitivity in old and young cells, a narrower range of HU was examined for the second round of pin stamping. The concentrations of HU used in this experiment were 0, 10, 20, 30, 40, and 50 mM. Again no distinction could be drawn between the HU sensitivity of old cells to the HU sensitivity of young cells. A closer look at concentrations between 0 to 10 mM and 100 to 300 mM may show the differences we are looking for. The increase resistance to HU of sml1 was not shown; therefore, there needs to be a tighter range of concentrations between 100 and 300 mM. It is possible that old cells are more resistant to HU because they are up regulating RNR. Double Strand Breaks Do Not Increase in Old Cells Replication slow zones are regions of DNA within the chromosomes at which replication forks slow down. These zones were first discovered in MEC1 mutants, where the slow zones turned into double stranded breaks (DSB) (95). Since Mec1p is the sensor for stalled replication forks, MEC1 mutants cannot fix stalled forks. In MEC1 mutants, replication forks spend more time at the slow zones. This leads to more DSBs in these replication slow zones. In old cells, the diminishing amounts of dNTPs caused by the episomal replication burden could cause replication forks to stall more frequently and for longer periods of time. This would in turn lead to more DSB. To test whether magnetically sorted old cells have more DSB then young cells, pulse field gels were used to separate chromosomes. After the gel was completed, it was transferred to positively charged nylon and probed with the CHA1 probe. CHA1 is located on one end chromosome III (95). If a DSB occurred, then faint bands should appear below the chromosome III band. These bands are shortened versions of chromosome III. No discernable difference could

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49 be observed between young and old cells. There were no shorter bands on the blot; therefore, there were no DSBs. The original paper describing DSBs in mec1 mutants used a synchronized population of cells. DNA was extracted from cells in the process of S phase. By using cell synchronization, DSB formation in old cell might be able to be seen. Since there is no positive control ( mec1 synchronized) in Figure 4-5, it is hard to say that the absence of DSB detection conclusively shows that DSB do not form in old cells. Figure 4-5. Southern of DSB in yAF6 (WT), W1488-4C (WT), sml1 mec1 sml1 and rad53 sml1 Pulse field gel was run according to materials and methods. Southern was transferred to positively charged nylon under alkaline conditions. The blot was probed with the CHA1 probe described in materials and methods. Phosphorylation of Rad53p Does Not Increase in Old Cells The protein that senses DSB and stalled replication forks in the cell, Mec1p, phosphorylates Rad53p (96) If Rad53p phosphorylation level increases in old cells, then this pathway could be implemented in aging. After magnetically sorting old cells, protein lysates were extracted. Several controls were also completed in parallel. HU causes a

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50 signal cascade through this DNA repair pathway resulting in the phosphorylation of Rad53p and the release of the RNR complex by Sml1p. The phosphorylation can be seen by a shift upward of Rad53p band on a gel (96). A rad53 sml1 strain was added to show which band is the Rad53p (the one not present in this strain). A mecl sml1 strain was used to show a less phosphorylated Rad53p. The western blot shows that there was no increase in the phosphrylation levels of Rad53p between young, old, and older (double magnetic sort) cells. Even between cells that have have a variety of ERC levels ( fob1 WT, and sir2 ), there was no difference in the phosphorylation of Rad53p. Figure 4-6. Rad53p phosphorylation in young and old cells. The upper dispersed band in the W1488-4C (+HU) is the phosphoralated version of Rad53p. The lower tight band in -HU lane of W1488-4C is the unphosphorylted state of Rad53p. Since this is a polyclonal antibody, there is some nonspecific binding to bands still present in the rad53 sml1 (lower band in the blot). ERC Competition with a CEN Plasmid Throughout Yeast Life Span By transforming pAF32 into WT, sir2 and fob1 strains the effects of ERCs on a CEN plasmid ccan be seen. These strains have different levels of ERCs and in turn different life spans. Sir2 produces the most ERCs. WT produce slightly less. Fob1 produces very few. pAF32 is a stable plasmid that contains a CEN element. It represents licensing of a single origin during the accumulation of ERCs. If ERCs cause an episomal replication burden by abnormal licensing of ARSs throughout the genome, then pAF32 should fail to replicate more often in strains with more ERCs.

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51 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 10 20 30 40 50 Generations Fraction of Cells Viable W303R5 sir2 fob1 Figure 4-7. Life span of W303R5 (WT), sir2 and fob1 during the CEN loss experiment. W303R5 had a mean life span of 22.4 and an n = 29. Sir2 had a mean life span of 12.6 and an n = 28. Fob1 had a mean life span of 30.1 and an n = 30. We followed the inheritance pattern of pAF32 throughout the life span of the various transformants. Because pAF32 contains the ADE2 gene, its inheritance can be followed by colony color (Appendix I-K). After the pedigree analysis was completed, the life spans of the three strains (with a CEN plasmid) were consistent with the life spans that are already published in the literature (without a CEN plasmid) (Figure 4-7). After a close analysis of the pedigrees, they show that the sir2 stain lost the plasmid at earlier divisions than the both WT and fob1 strains. WT, which has intermediate number of

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52 ERC, had intermediate loss of pAF32. The fob1 strain had the fewest ERCs and the latest lost of the CEN plasmid (Figure 4-8). While there was a low n, this result suggests that ERCs inhibit CEN plasmid replication. It also says that episomes may cause aging in old cells by sequestering needed replication factors away from the genome. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 10 20 30 Generation at which CEN Plasmid was lost Fraction of Cells W303R5 sir2 fob1 Figure 4-8. The age which WT, sir2 and fob1 lose plasmid. CEN plasmids were lost at a mean generation time of 11.4, 6.25, and 14.3 (respectively). The number of cells analyzed in each strain were n = 8, n = 4, and n = 7 (respectively). Mitotic Stabilities in the Presence of ERCs To further investigate ERCs role in the cell, mitotic stabilities were examined. A change in mitotic stability of ARS plasmids may be expected in strains with various

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53 levels of ERCs accumulation. Fob1 WT, and sir2 strains were tested with pAF31 (ARS), pAF32 (CEN), pJPA133 (ARS), and pJPA136 (CEN). The two sets of plasmids were used to control for the use of the ADE2 or LEU2 selectable markers. The sir2 strain had an increased mitotic stability of pAF31. Normally pAF31 has a mitotic 0 10 20 30 40 50 60 70 80 90 100 W303 pAF31 fob1 pAF31 sir2 pAF31 W303 pAF32 fob1 pAF32 sir2 pAF32 Strain/Plasmid Mitotic Stability n = 7 9 11 13 13 14 Figure 4-9. Mitotic stabilities of pAF31 and pAF32. The two plasmids were tested in strains with various amounts of ERCs. stability of 16% (W303). In the sir2 strain, pAF31 had a mitotic stability of 29%. The sir2 strain had a slight increase in the mitotic stability of pJPA133, but it was not significant. The mitotic stability went from 17% (W303) to 21% ( sir2 ), but these

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54 numbers fell within the standard deviation of each other. This could be explained by the smaller size of pJPA133 from that of pAF31. Very small plasmids are known to have different mitotic stability characteristics. The ARS plasmid, pAF31, may have reached a critical threshold in size for ERCs to play a role in mitotic stability. The fob1 strains inability to further reduce the mitotic stability of pAF31 shows that there may be a critical mass of episomes a cell can handle. If the increase in ERCs from fob1 to W303 was not enough to change mitotic stability, then the decrease in size from pAF31 to pJPA133 could be not enough to change mitotic stability. 0 10 20 30 40 50 60 70 80 90 100 W303 pJPA133 fob1 pJPA133 sir2 pJPA133 W303 pJPA136 fob1 pJPA136 sir2 pJPA136 Strain/Plasmid Mitotic Stability Figure 4-10. Mitotic stabilities of pJPA133 and pJPA136. The two plasmids were tested in strains with various amounts of ERCs.

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55 Plasmid Accumulation in sir2 and fob1 Strains Again fob1 sir2 and wt strains were transformed with pJPA133 and pJPA136. Magnetic sorts were completed to obtain young and old cells. The episomal replication burden model predicts that accumulating plasmid will compete. In this example, ERCs will compete with pJPA133 for the replication machinery. The accumulation of the ARS plasmid will decrease in the strains with increased ERC production. Figure 4-11. Southern of showing plasmid competition phenomenon. XhoI digested genomic DNA yields a 8.935 kb LEU2 band. Other bands are plasmid derived. Southerns were completed on the magnetic sorts to quantify the copy number of the plasmids in each strain. As predicted old sir2 cells which contains more ERCs, had fewer ARS plasmids (pJPA133) per cell (165 plasmids/cell). WT had an intermediate number (272 plasmids/cell), and fob1 with the fewest number of ERCs contains the most copies of the plasmid (293 plasmids/cell) in old cells. pJPA133 also had a different copy number in the young strains. The copy numbers were 6, 4.6, and 2.8 with respect to

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56 fob1 WT, and sir2 This again mimicked the results from the old cells, but to a lesser degree. Young and old cells of all of the strains contained relatively the same number of pJPA136 (CEN). This argues for a competition between ARS episomes for replication machinery in the cell. To further extrapolate this idea, episomes may also compete with the genome for the replication machinery and cause aging. Another explanation of this result may be that there is a critical mass of episomes allowed in the nucleus. After that mass is reached episomes begin to be pushed out. 0 50 100 150 200 250 300 350 sir2 pJPA133 wt pJPA133 fob1 pJPA133 sir2 pJPA136 wt pJPA136 fob1 pJPA136 Strains Plasmid Copy Number per Cell Young Old Figure 4-12. Quantitation of plasmid competition Southern. Quantitaion was completed on the blot in Figure 4-11 by a Phosphuor imager. Summary The mechanism by which episomes cause aging has been very illusive. In this chapter several experiments have been designed to tease out the mechanism. Although some experiments gave negative answers, they did give definitive answers. While the

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57 exact mechanism of episomal aging is still not well known, the experiments strongly suggest in what direction subsequent experiments should go. The major experiments in this chapter show a few things: CEN plasmids are lost at younger generation times in strains with more ERCs. The mitotic stability of ARS plasmids increases in strains with more ERCs. Old cells of strains with more ERCs accumulate fewer ARS plasmids. Together they say that as episomes accumulate there is an increasing strain on the cells replication machinery.

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58 CHAPTER 5 CHROMATIN SILENCING AND EPISOME FORMATION The major role of Sir2p in aging is its ability to deacetylate histones. By deacetylating histones in the rDNA, it is silencing the DNA. More specifically it is compacting the chromatin and making it less accessible. This results in fewer ERCs produced and in turn a longer life span. Strains that over express Sir2p have fewer ERCs and live longer. Conversely SIR2 deletions create more ERCs and have a shorter life span because it cannot deacetylate histones. If the histone acetylation status in the rDNA is central in ERC aging, then limiting a cells ability to acetylate histones could be as important as Sir2p ability to deacetylate histones. Histone acetyl transferases (HATs) are the enzymes that acetylate histones. In addition to the enzymes responsible for the acetylation and deacetylation of histones the pool of acetyl CoA may be important to aging. Acetyl CoA is the substrate for HATs in the acetylation of histones. A closer look at the production of acetyl CoA may lead to mutants that increase life span. These ideas are illustrated in Figure 5-1. Two enzymes are responsible for the production of acetyl CoA, Acs1p and Acs2p (Acetyl CoA Synthetase). Acs1p has a K m 30 times lower than Acs2p (97). This does not mean that it does most of the conversion of acetate to acetyl CoA. The two enzymes are regulated very differently. Acs1p is completely repressed by glucose (100 mg/L), up regulated in ethanol, and further increased in acetate medium. Where as Acs2p is maintained at a constant level in glucose and ethanol, but acts sporadically in acetate (97). An ACS2 deletion cannot grow on glucose as a carbon source. This is because of

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59 the repression of ACS1 by glucose. If ACS2 is deleted and ACS1 is repressed by glucose, then there is insufficient ACS activity in the cell, and the cell cannot survive. The cell needs acetyl CoA to survive. Figure 5-1. The acetylation of histones and its affect on ERC production and life span. ACS2 and ACS1 is Required for Normal Life Span Deletions of ACS1 and ACS2 were obtained from the Clusius lab. It was predicted that deletions of the ACS genes would lead to an extended life span. This is because acetyl CoA levels in the cell would drop. Histones would be acetylated less, and the production of ERCs would decrease causing a longer life span.

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60 Life spans were completed on both glucose and ethanol. Surprisingly T23D, the strain background, has a much longer life span than the W303R5 derivatives that have been used in previous studies throughout this dissertation and other strains in published papers. Never the less, there was a reduction in life span in acs1 from the controls. Interestingly acs2 had an even shorter life span than asc1 (Figure 5-2). These reductions in life span are opposite of what one might conclude, from the model described in Figure 5-1. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 20 40 60 80 Generations Fraction of Cells Viable T23D E 621 E 625 E T23D D 621 D Figure 5-2. Life span of ACS deletions. T23D (control) on ethanol (E) had a mean life span of 55.2 and an n=59. 621 ( acs1 ) on ethanol (E) had a mean life span of 47.4 and an n=59. 625 ( acs2 ) on ethanol (E) had a mean life span of 16.6 and an n=51. T23D (control) on glucose (G) had a mean life span of 35.0 and an n=59. 621 ( acs1 ) on glucose (G) had a mean life span of 24.8 and an n=59.

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61 A closer look at metabolism shows that acetyl CoA is used for many processes in the cell besides acetylation of histones. Acetyl CoA is one of the entrance points into the Citric Acid Cycle. It is also used in lipid synthesis. ACS2 is known to be coregulated with structural genes of fatty acid biosynthesis because of an upstream ICRE (inositol/choline-response element). These and other pathways may be more important to a cells health than the predicted decrease in the production of ERCs by the ACS deletions. In addition to looking at the affects of Acs1p and Acs2p in a cell, we inadvertently observed a difference in life span between cells grown on glucose and cells grown on ethanol as a carbon source. When T23D was grown on ethanol it had a mean life span of 55.2 and a maximum life span of 77 Generations. This is longer than the longest life span we have seen published in the literature. To our knowledge the longest life span published is a mean life span of 36.1 and a maximum life span of 74 (98) ACS2 Increases ERC Production To further explore ACS1 and ACS2 s role in aging, ERC production and accumulation was measure in old and young cells of the ACS deletions. According to the model illustrated in Figure 5-1, ACS deletions should lead to a decrease in ERC production. Cells were grown in ethanol and then age fractionated by magnetic sorting. T23D (control) had very few to no ERCs in the old cells. This may be why it has a longer life span than W303R5. What is the genetic difference between T23D and W303R5. T23D is a diploid strain while W303R5 is a haploid. Previous experiments have shown that the ploidy of a strain is not important to life span (99). Another difference is that W303R5 is ade2 his3 leu2 trp1 and ura3 ; and T23D has no

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62 auxotrophic markers. Some of these genes can be compared by life spans that were completely separately, and they do not seem to influence life span. Interestingly, the ACS2 deletion produces more ERCs. While the ACS1 deletion does not produce ERCs. Acs2 s ERC production may attribute to its shortened life span. Figure 5-3. Sorts of young and old ACS deletion strains. The southern blot was probe for rDNA. Light banding in the old acs2 sample can be observed at a high molecular weight. These are ERCs. Summary ACS1 ACS2 ethanol as a carbon source, and fatty acid biosynthesis are all linked to aging. Further exploration is needed to fully understand how acetyl CoA is involved in

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63 aging. Another noteworthy result is that T23D lives longer than W303R5. It would be interesting to know what genetic differences contribute to its extended life span. Many experiments could easily be designed to determine the genes that help it live longer.

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64 CHAPTER 6 LOOKING AT POSSIBLE MECHANISM OF CELLULAR AGING YCA1 and Apoptosis in Yeast Apoptosis and aging are intimately linked. Since programmed cell death, apoptosis, contributes to the aging of metazoans, looking at a known yeast caspase, an apoptotic regulator, seemed reasonable. Yca1p is the only known caspase like protein in yeast (100) It has been shown to be required for hydrogen peroxide induced apoptosis. When YCA1 is deleted, it increases yeast chronological life span (100). To further investigate the role of Yca1p in the aging process we constructed a YCA1 deletion in our W303AR5 strain. It was created by using microhomology to YCA1 and inserting HIS3 inside of the gene (See Materials and Methods). After the insertion of HIS3 into YCA1 we confirmed by southern that the constructed W303AR5 yca1 :: HIS3 strain was correct. A control strain was also created by repairing the his3 locus of the W303AR5 strain. This was to insure that the HIS3 status of the cell would not contribute to a lifespan change. The two strains were compared through replicative life spans (Figure 6-1). There was no statistical difference between the yca1 deletion and the control strain. The interpretation is that while yca1 is necessary for a normal chronological life span, it is not required for replicative life span. This experiment illustrates the fundamental difference between a chronological and replicative life span. A chronological life span demonstrates a cells resilience and ability to replicate after being in a saturated culture full of cells and depleted of nutrients. In a replicative life span, cells are spaced out very far from each other, so it is unlikely that they will run out of nutrients. It is a measure of

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65 a cell replicative potential, not its resilience over time. It is a subtle difference, but important when trying to address different questions about cellular aging. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 10 20 30 40 Generations Fraction of Cells Viable Control yca1 Figure 6-1. Life span of yca1 This life span was completed on rich media (YPD). The control strain had a mean life span of 22.1 (n=60). The yca1 deletion had a mean life span of 21.4 (n=57). Shu Gene Family and Mutation Suppression in Aging Using a CAN1 forward-mutation assay, the SHU (sensitivity to hydroxyurea) genes were discovered. These genes are required to prevent spontaneous mutations. In the assay, 4,847 yeast deletion mutants (from the yeast deletion project) were screened for the ability to spontaneously become canavanine resistant ( can1 ) (101) In collaboration with the laboratory of Rodeny Rothstein, four of the genes discovered in this screen ( SHU1 SHU2 SHU3 and CSM2 ) were looked at by replicative

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66 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 10 20 30 40 Generations Fraction of Cells Viable W303R5 shu1 shu2 shu3 csm2 4X Figure 6-2. SHU genes role in life span. The mean life span of W303R5 is 24.5 (n=60). The mean life span of shu1 is 25.3 (n=60). The mean life span of shu2 is 25.8 (n=58). The mean life span of shu3 is 23.2 (n=56). The mean life span of csm2 is 22.5 (n=59). The mean life span of the quadruple deletion ( shu1 shu2 shu3 and csm2 ) is 21.5 (n=59). life span. They are believed to be involved in DNA replication. These experiments were completed in the Rothstein lab. These genes ability to suppress mutations and their involvement in DNA replication make them a candidate for having an increased life span. The Rothstein lab had preliminary data that suggested this with one or more of the mutants. After the experiment was completed all strains had very similar life spans (Figure 6-2). A close look at the p-values obtained from the Wilcoxon two-sample paired

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67 signed rank test shows that there may be a small difference between the strains (Table 61). The experiment needs to be repeated (one of the few that has not been) before any major conclusions can be drawn. Table 6-1. P-values of the SHU deletion life spans. Comparison #1 Comparison #2 P-Value W303R5 shu1 0.032 W303R5 shu2 0.003 W303R5 shu3 1.22 x 10 -7 W303R5 csm2 9.07 x 10 -6 W303R5 Quadruple 4.50 x 10 -8 shu1 shu2 0.090 shu1 shu3 1.14 x 10 -4 shu1 csm2 2.12 x 10 -6 shu1 Quadruple 3.30 x 10 -8 shu2 shu3 1.47 x 10 -4 shu2 csm2 1.02 x 10 -5 shu2 Quadruple 4.23 x 10 -6 shu3 csm2 0.016 shu3 Quadruple 1.68 x10 -5 csm2 Quadruple 0.002 Summary This chapter focuses on ideas that were not specifically linked to our episomal aging model. Because these concepts could have played a role in the broader scope of aging, we explored them. It is now known that YCA1 does not affect replicative life span.

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68 We also know that more experiments need to be completed to show definitively the subtle difference between the SHU strains

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69 CHAPTER 7 DISCUSSION Budding yeast is an excellent system in which to study cell-autonomous mechanisms of aging. Mechanisms linked to genome stability, metabolic damage, and metabolic regulation have been found to regulate yeast replicative life span (4547,52,53,102). Sinclair and Guarente have proposed that a key regulator of life span is the cellular level of extrachromosomal rDNA circles ( E R C s) (54). To study this proposal, we have used plasmids to model E R C inheritance and accumulation, two processes that govern E R C levels in yeast cells. Our work shows that plasmid DNAs bring about significant reductions in yeast life span. We find that ARS1 and 2 origin plasmids specifically accumulate in old yeast cells, and that the level of accumulation of ARS1 and 2 origin plasmids in old cells correlates with the extent of reduction in life span. This is the first demonstration to our knowledge of an inverse relationship between DNA episome level in old cells and reduction in life span. We find that plasmids have a direct effect on life span and do not indirectly reduce life span by increasing recombination at the rDNA locus and increasing E R C levels in transformed cells. Analysis of the terminal morphology of senescent cells indicates that plasmids do not cause a stochastic arrest in the cell cycle, which is consistent with a normal aging process. Reduction in life span does not require that plasmids carry rDNA repeat sequences, and the presence of a full-length, functional 9.1 kb rDNA repeat on a plasmid does not augment reduction in life span. These findings confirm the work of Sinclair and Guarente (54), and provide significant new support for their E R C model by directly

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70 demonstrating a relationship between plasmid inheritance, plasmid accumulation, and reduction in life span. Our studies also highlight the value of plasmids as tools to investigate properties of E R C s that are relevant to the aging process in yeast. Why Do ARS Plasmids Accumulate in Mother Cells? It has long been appreciated that ARS plasmids are inherited asymmetrically and accumulate in mother cells (43). This accounts for the high copy number and low mitotic stability of ARS plasmids. However, accumulation of ARS plasmids in cells that are multiple generations old has not been directly demonstrated. Our studies are the first to directly demonstrate that ARS1 -containing plasmids accumulate to high levels in old yeast cells. Although ARS1 plasmid partitioning bias is well known, little is understood about its underlying mechanism. One possibility is that plasmid partitioning bias is due to the nature of cell and nuclear division in budding yeast. During closed mitosis in yeast, an intact nucleus elongates along the axis of the mitotic spindle and adopts an elongated dumb-bell shape due to constriction of the nucleus at the bud neck. Chromosomes pass though the constriction at the bud neck by virtue of their attachment to the mitotic spindle, which is able to exert force on chromosomes. In the absence of spindle attachment, passage of DNA molecules through the constriction at the bud neck may be limited. Consistent with this notion, the relatively small (1.45 kb) TRP RI plasmid has been shown to be inherited efficiently and to exhibit high mitotic stability (103). The small size of the TRP RI plasmid may allow it to readily distribute between mother and daughter cells through the bud neck constriction. Commonly used yeast recombinant DNA vectors are typically larger than the TRP RI plasmid and require cis acting sequences and trans -acting factors to be stably inherited.

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71 Why Do Budding Yeast Exhibit a Mother Cell Plasmid Segregation Bias? One possibility is that mother cell segregation bias is a mechanism to protect progeny cells from potential parasitic effects of episomal DNAs acquired from the environment. The 2 circle is a commensal episomal DNA that Futcher has depicted as a sexually transmitted selfish DNA (89) The 2 circle depends on its capacity to overcome mother cell segregation bias (see below) in order to survive in a host population in the absence of any selective value. Another possibility is that mother cell segregation bias is a mechanism to increase the longevity of progeny cells by limiting transmission of E R C s. Why Do ARS1 Plasmids Bring About Cellular Senescence More Rapidly than Do E R C s? One possibility is that virgin mothers contain more plasmids than E R C s at the start of life span experiments. Virgin mothers must contain at least one ARS plasmid, but probably contain on average ~0.5 E R C per cell. The difference in origin strength between ARS1 and the rDNA ARS may also be important. ARS1 is a relatively strong ARS, and capable of supporting rapid plasmid accumulation in mother cells. E R C s contain a comparatively weak ARS that is likely to support only relatively slow accumulation in mother cells. The rDNA ARS contains an ACS (ARS consensus sequence) that departs from the consensus at position 1 a change that has been shown to reduce ARS function, primarily by limiting DNA unwinding (81). This difference in strength could explain why ARS1 plasmids bring about senescence in mother cells more rapidly than do E R C s. ARS1 plasmids are replicated more efficiently than E R C s, which increases the rate of ARS1 plasmid accumulation in mother cells compared to E R C s

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72 Do Cis -acting Sequences that Counteract Mother Cell Segregation Bias Suppress Reduction in Life Span by ARS1 Plasmids? Yes, ARS1/CEN4 plasmids reduce life span to a lesser extent than ARS1 plasmids, which is consistent with results of Sinclair and Guarente (54). However, inclusion of CEN4 on ARS1 plasmids suppresses the reduction in maximum life span by ARS1 plasmids, but does not fully suppress the reduction in average life span. Our studies also directly show that ARS1/CEN4 plasmids do not accumulate in ~7 generation old mother cells. The reduction in life span is not specific for the combination of ARS1 and CEN4 The combination of ARSH4 and CEN6 (in pRS314, (77)) reduces average life span with a minimal effect on maximum life span. The fact that centromeric DNA elements suppress reduction in maximum life span supports the conclusion that ARS1 plasmids exert their effect by accumulation in mother cells, as discussed above. Do 2 Micron Circles Reduce Life Span? It is initially surprising that cir 0 cells did not have an increased life span compared to cir + cells, especially since 2 origin plasmids accumulated in old cells. Quickly we realized that the 2 circles and 2 origin plasmids were very different. 2 circles did not accumulate in old cells and 2 origin plasmids did. With this knowledge of 2 accumulation, it becomes obvious that 2 circles would not decrease life span. Why Do 2 Micron Origin Plasmids Reduce Life Span? Although both 2 origin plasmids and 2 circles contain the REP3/STB cis -acting stability element, 2 origin plasmids contain a single 599 bp segment, whereas 2 circles contain two 599 bp segments arranged as an inverted repeat (76,80). More efficient autoregulation of 2 circle copy number and inheritance is likely to prevent accumulation in old cells. It is important to note that 2 circles can be toxic to cells

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73 when present at high copy number. Constitutive expression of the 2 amplification machinery results in high copy number and has deleterious effects on cell growth (76). Similarly, mutations in NIB1/ULP1 result in unusually high levels of 2 circles, formation of large inviable or mitotically arrested cells, and clonal lethality (104). Studies by Dobson and coworkers indicate that an abnormal form of Rep2p, a 2 circleencoded plasmid partitioning protein, accumulates in ulp1 mutants, suggesting that ULP1 is involved in partitioning of 2 circles during mitosis (M. Dobson, personal communication). This suggests that high levels of 2 circles in nib1/ulp1 mutants may result from asymmetric inheritance. In this sense, phenotypes associated with nib1 / ulp1 defects may share mechanistic underpinnings with senescent phenotypes associated with asymmetric inheritance of plasmids and E R C s. Why Do 2 Micron Origin Plasmids Have an Intermediate Effect on Life Span? Although 2 origin plasmids accumulate in ~7 generation old mother cells, they attain levels approximately half that observed with ARS1 plasmids. As mentioned above, comparison of results with 2 origin and ARS1 plasmids supports an important semiquantitative inverse relationship: the extent of plasmid accumulation in old cells correlates with the extent of reduction in life span. Why Does Transformation with pJPA114 Lead to 2 Micron Circle Loss? Recent studies of 2 micron plasmid partitioning have made great strides in revealing roles for cis-acting elements and trans-acting factors in substantial cellular and molecular detail (105,106). These studies and earlier studies (76,87) suggest that inheritance of 2 micron plasmids has little mechanistic overlap with inheritance of replicating (ARS) plasmids such as pJPA114. Thus, it seems unlikely that pJPA114 competes with 2 micron circle for a limiting amount of (a) specific mitotic partitioning

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74 factor(s). Another possibility is that pJPA114 adversely affects 2 micron circle copy number, which in turn adversely affects transmission to daughter cells. We have observed that 2 micron circle DNA levels are reduced 30-40% in ~7-generation old cells containing yeast replicating plasmid pJPA113, but not in ~7-generation control cells lacking pJPA113. pJPA113 accumulates to high levels in ~7-generation old cells (86), and perhaps 2 micron circle DNA levels are reduced as a result of this accumulation. pJPA114 attains a high copy number in young cells (86), and is likely to accumulate in old cells, like pJPA113. Although these findings are not conclusive, they are consistent with competition between pJPA114 and 2 micron circles for DNA replication factors and/or precursors, which could lead to reduced 2 micron circle copy number and impaired transmission to daughter cells. By What Mechanism(s) Do Plasmids, and by Implication E R C s, Reduce Life Span in Yeast? It is clear that asymmetric inheritance of plasmid DNAs has the potential to burden mother cells with high DNA content. If we assume that a 5 kb plasmid is replicated once each S phase, and uniformly inherited by the mother cell during M phase, then 12 doublings will yield a plasmid DNA content in excess of the nuclear genomic DNA content (5 X 2 12 = 20.5 Mb plasmid DNA content > ~13 Mb nuclear genomic DNA content). Of course, this example is an oversimplification and omits factors such as origin firing frequency and segregation efficiency. However, we note that after 12 generations, 90% of pJPA113 (5.7 kb ARS1 plasmid) transformants were senescent and after 20 generations, 90% of pJPA133 (4.8 kb ARS1 plasmid) transformants were senescent. The fact that significant percentages of senescent mother cells arise between 10 and 20 generations is consistent with the accumulation of plasmid DNA content to a

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75 level that approaches or exceeds nuclear genomic DNA content. Similarly, Sinclair and Guarente have estimated that the E R C content of old cells exceeds the content of the linear genome (54). Why Are There Less Plasmid Accumulation in Strains that Produce More ERCs? This concept of plasmid competition brings us to believe that there is a replication burden in old cells. Two things could cause the loss of pAF32 (CEN), when it is in the presence of ERCs. A limiting replication factor or DNA substrate could be soaked up by the large quantity of ERCs and not allow the single copy pAF32 to replicate. In another scenario the ERCs could act almost like a physical barrier making it harder for the plasmid to leave the cell. Inheritance and replication are the two mechanisms that are central to the characteristics and behavior of plasmids. The mitotic stability in sir2 WT, and fob1 eliminates the concept of ERCs acting as a physical barrier for inhertance of pAF32. It also raises new questions when looking at the ARS plasmids (pAF31 and pJPA133). Returning to the two plasmid processes, ERCs could be increasing plasmid inheritance or replication. While it is unlikely that ERCs are causing an increase in replication, a look at inheritance allows us to start constructing some models. It is possible that there is a limited amount of space in the nucleus. The total number of episomes cannot be higher than some critical mass of DNA. This would cause the two accumulating episomes to be pushed out of cells and inherited better. Saturation of the plasmid bias machinery could be another mechanism that increases mitotic stability. Episome accumulation in old cells of strains that produce various levels of ERCs shows us that plasmid competition is very real. The reduced level of pJPA133 in sir2 is about half of the copy number than in W303R5. This is dramatic. The most important

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76 part of the plasmid competition phenomenon is whether, it is an output of how episomes causes aging. Mechanistically these two ideas could be very similar. If this is true, we will be able utilize the versatility of plasmids to discover how ERCs cause aging. Is There Episomal Aging in Metazoans? While ERCs have not been found in metazoans, it is hard to say that the absence of evidence for them proves there is no episomal aging is higher organisms. It is possible that another highly repetitive sequence can recombine to form episomes, but they have been very difficult to detect. Also, DNA viruses could be interpreted as episomes that reach high copy within a cell. An analogy can be draw between ERC replication stress and viral commandeering of a cells replication machinery. Both may lead to problems during DNA replication and hence genomic instability. Genomic instability could facilitate the mutations and recombination in various cancer causing genes and increase the incidence of cancer.

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77 CHAPTER 8 MATERIALS AND METHODS This chapter contains the methods and procedure used for experiments throughout this dissertation. Yeast Strains and Plasmids W303AR5 ( MAT a leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 ADE2::rDNA [cir + ], (54)) was obtained from D. A. Sinclair. yAF5 and yAF6 were constructed by integrating linearized pRS305 and pRS306 (77), respectively, into the leu2-3,112 or ura3-1 loci of W303AR5, respectively, and genotypes were confirmed by Southern blotting. Plasmids were transformed into W303AR5 using a standard lithium acetate method (107). All experiments were done with freshly-prepared, independentlyisolated, colony-purified transformants. Unless otherwise noted, yeast were grown on selective SD drop in medium (88). Descriptions of plasmids are provided in Table 1. A 200 bp fragment containing ARS1 was amplified by P C R with primers 5-GGAAGCTTCCAAATGATTTAGCATTATC-3 and 5-CCGAATTCTGTGGAGACAAATGGTG3 using template YRp17. A 200 bp fragment containing the rDNA ARS was amplified by P C R with primers 5-CCAAGCTTGTGGACAGAGGAAAAGG -3 and 5-GGGAATTCATAACAGGAAAGTAACATCC -3 using template pJPA102 (rDNA repeat with AhdI endpoints in pCR4, see below). A 753 bp fragment containing CEN4 was amplified by P C R with primers 5-GCGGATCCCCTAGGTTATCTATGCTG -3 and 5-GGGAATTCCTAGGTACCTAAATCCTC-3 using template YCp50. A 1346 bp region of 2 circle DNA, containing the REP3/STB cis

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78 acting stability element and a single 599 bp repeat region, was amplified by P C R with primers 5-CCGGATCCAACGAAGCATCTGTGCTTC -3 and 5-CCAAGCTTTATGATCCAATATCAAAGG -3 using pRS424 as template. rDNA repeats were amplified by P C R using as template size-selected (8-10 kb), genomic DNA that was digested with the appropriate enzyme (AhdI, PsiI, or XmaI). The following primer pairs were used: AhdI endpoints, 5-GGGATCCATGTCGGCGGCAGTATTG-3 and 5-CCTGCAGCTGTCCCACATACTAAATCTCTTC-3 ; PsiI endpoints, 5-GGGATCCTAATATACGATGAGGATGATAGTG3 and 5-CCTGCAGTAATAGATATATACAATACATGTTTTTACC-3 ; XmaI endpoints, 5-CCCGGGGCACCTGTCACTTTGG-3 and 5CCCGGGTAAACCCAGTTCCTCACTAT-3 P C R was performed for 20 cycles with 15-second denaturation and annealing times using PfuTurbo DNA polymerase (Stratagene). P C R products were purified (Qiagen), digested with restriction enzymes and ligated directly into recipient vectors, or cloned into pCR4TOPO (Invitrogen), excised, gel-purified, and ligated into recipient vectors (see Table 21). ARS elements were cloned between HindIII and EcoRI sites. CEN4 was cloned between EcoRI and BamHI sites. The 2 origin was cloned between HindIII and BamHI sites. rDNA inserts were cloned between PstI and BamHI sites in pAF15, which is derived from pRS424 and contains loxP sites that were inserted at EcoRI and SpeI sites using annealed primer pairs: 5-AATTATAACTTCGTATAATGTATGCTATACGAAGTTAT3 and 5-AATTATAACTTCGTATAGCATACATTATACGAAGTTAT -3 (EcoRI); 5-CTAGATAACTTCGTATAATGTATGCTATACGAAGTTAT -3 and 5-CTAGATAACTTCGTATAGCATACATTATACGAAGTTAT-3 (SpeI). All cloned inserts were sequenced in their entirety. Plasmids pJPA105, pJPA106, and pJPA107 (that contain

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79 rDNA inserts) were propagated in E. coli DH5a grown in LB media with 25 g/ml carbenicillin at 30C to avoid insert instability. Mitotic Stability For each plasmid, five transformants were grown in selective SD liquid medium for 2 days at 30C to saturation (OD 600 = 1.1-1.5; 0.5-1X10 7 cfu/ml; growth to late log gave results similar to stationary phase). Approximately 200-250 colony forming units (cfu) of each transformant were plated on non-selective SD medium, grown for 2-3 days at 30C, replica plated onto selective and non-selective agar media, and grown for 3-4 days at 30C. After these plates grow the total number of colonies that grew under no selection are counted from the first plate. The number of colonies that grew under selection are counted from the second plate. Dividing the number of colonies from the second plate by the number of colonies on the first plate gets the percent of cells in the population that had the plasmid. This is the mitotic stability. It is simply the number of colonies that contained the plasmid divided by the total number of colonies. Replicative Life Span Determinations Replicative life span determinations were done essentially as described (92) with a few modifications. Six transformants were streaked individually on one side of an SD agar plate, and 10 virgin mother cells from each (n=60) were positioned in an orthogonal grid pattern. Virgin mothers that failed to give rise to 5 daughters were not included in the data set. Due to the low mitotic stability of ARS-plasmids, it was necessary to start with approximately 250 virgin mother cells from ARS1 -plasmid transformants to obtain n=50-60 for life span determinations. A Zeiss Tetrad microscope equipped with 16X eyepieces was used for micromanipulations as described (88). SD agar plates were weighed at the beginning of each experiment and sterile water was pipetted into four

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80 small notches at the edge of each plate on a daily basis to compensate for evaporation and prevent increases in osmolality, which could potentially affect results (108). During life span experiments, plates were incubated at 30C during the daytime and stored overnight (~12 hours) at 14C. We found that extended periods ( # 24 hours) at 14C reduced life spans of transformants and control strains (data not shown). At the end of a life span experiment, mother cells not having divided for 2 days were transferred to non-selective SD or YPD medium, and cells that resumed mitosis were excluded from the data set. This allowed us to exclude data from mother cells that stopped dividing due to plasmid loss rather than due to cell senescence. Data were entered into an Excel spreadsheet template file (available on request) that automatically calculated relevant life span data values and performed Wilcoxon two-sample paired signed rank tests. Images of terminal cells were collected using a Spot-2 CCD camera (Diagnostic Imaging) affixed to a Zeiss Tetrad microscope and terminal cell morphology analysis was done as described (70) Southern Blot Analysis and Quantitation DNA was extracted from yeast cells using a glass beads/phenol method, digested with restriction enzymes according to the supplier (New England Biolabs), separated on 0.8% agarose gels (200 V/hours), and capillary transferred to positively-charged nylon membrane under alkaline conditions using standard methods (109). For each plasmid copy number and E R C monomer level determination, five plasmid transformants were analyzed in parallel. Digestion with BamHI or PstI yielded single plasmid-specific or genome-specific bands of different sizes that hybridized to 32 P-labeled probe generated by random-primed labeling (New England Biolabs). PstI and BamHI do not cleave rDNA. Genomic bands were used as internal standards for measurements of plasmid levels. Chromosomal rDNA bands were used as internal standards for measurements of

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81 E R C monomer levels. Blots were hybridized first to URA3 or LEU2 probe, followed by stripping and hybridization to rDNA probe. Data from the same blots were used to prepare Figures 2 and 3B. Southern data were acquired with a Typhoon PhosphorImager and analyzed using ImageQuant software (Molecular Dynamics). Pulse field gel electrophoresis was completed at 14C with a Bio-Rad CHEF-DR II. 1% agarose gels were run in 0.5X TBE for 30 hours. The voltage used was 200V with a switch time starting at 5 seconds and ending at 30 seconds. Magnetic Cell Sorting At any given time 1 in 512 cells is an eight generation old cell. This is due to the nature of a doubling population. 1/2 of the cells are new, zero generation daughters. 1/2 of the cell remaining (1/4) are 1 generation old cells. 1/2 of the cell remaining (1/8) are 2 generation old cells. This continues until the number becomes increasingly smaller. To extract the small number of old cells from a large population of cells, magnetic sorting is used. 1x10 8 cells are grown up and labeled with biotin. They are then grown over night in 1 liter of liquid medium. Since new cell wall synthesis occurs at the bud of the emerging cell, no biotin is transferred to the newly divided daughters. This results in a large population of cells that have their oldest cells labeled with biotin and the young cells are not. The cells are then spun down and concentrated into a smaller volume. Strep-avidin coated magnetic beads are mixed with the cells for 2 hours at 4C. All of the subsequent steps are done in the cold to ensure that the cell do not continue to grow. The strong interaction between biotin and avidin allows the magnetic beads to bind to the old cells. The old cells are then pulled out of solution with a strong magnet and the young cells are washed away. Eight washes are used to ensure that the population acquired at

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82 the end of the experiment is homogeneously old. The old cell final product is ready for further use in other experiments. Budscar Histograms After a sort for old cells, a bud scar histogram is conducted to determine the age distribution of the cells collected. When a cell divides a bud scar ring is formed at the point of budding and separation. The bud scar can be stained with a fluorescent dye calcofluor white MR2. A small fraction of old and young cells are stained with calcofluor and the number of bud scars on 50 cells are recorded. This creates a histogram of the number of cells vs. the number of budscars. rDNA Recombination Assay The rDNA recombination assay is designed to quantitate the level of recombination at the rDNA locus. W303AR5 has an ADE2 gene within the rDNA locus. In the absence of the ADE2 gene colonies become red in color, while wild type ADE2 are white. This color phenotype allows the scoring of ade2 colonies to be very easy. Saturated liquid cultures were prepared from five transformants. They were diluted and spread on 15 cm selective SD agar plates containing 5 g/ml adenine hemisulfate and 5 g/ml histidine to enhance red color production. They are allowed to grow at 30C for 2-3 days. The plates are then placed at 4C for 1-2 days for the color to develop. The number of half sector colonies, colonies that are half red half white, are scored in comparison to the number of all white colonies. All other partially sectored or red colonies are ignored. The reason only half sector colonies are scored is because half sectored colonies are colonies that have lost the ADE2 marker on the first division in the colony formation. A completely red colony may have become red at any time in growing in the liquid culture.

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83 By looking at the first division to forming a colony, all of the data can be normalized to a single mitotic event.

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84 APPENDIX A STRAIN: W303AR5+pJPA113 MEDIA: SD aHLW Terminal morphology of senescent cells with its life span directly below the image.

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85 APPENDIX B STRAIN: W303AR5+pJPA116 MEDIA: SD aHLW Terminal morphology of senescent cells with its life span directly below the image.

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86 APPENDIX C STRAIN: W303AR5+pJPA138 MEDIA: SD aHLW Terminal morphology of senescent cells with its life span directly below the image.

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87 APPENDIX D STRAIN: yAF6 MEDIA: SD aHLW Terminal morphology of senescent cells with its life span directly below the image.

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88 APPENDIX E STRAIN: W303AR5+pJPA133 MEDIA: SD aHWu Terminal morphology of senescent cells with its life span directly below the image.

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89 APPENDIX F STRAIN: W303AR5+pJPA136 MEDIA: SD aHWu Terminal morphology of senescent cells with its life span directly below the image.

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90 APPENDIX G STRAIN: W303AR5+pJPA148 MEDIA: SD aHWu Terminal morphology of senescent cells with its life span directly below the image.

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91 APPENDIX H STRAIN: yAF5 MEDIA: SD aHWu Terminal morphology of senescent cells with its life span directly below the image.

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92 APPENDIX I STRAIN: W303R5 +pAF32 MEDIA: YPD Every two rows is the pedigree for a single cell.

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93 APPENDIX J STRAIN: FOB1 +pAF32 MEDIA: YPD Every two rows is the pedigree for a single cell.

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94 APPENDIX K STRAIN: SIR2 +pAF32 MEDIA: YPD Every two rows is the pedigree for a single cell.

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95 A PPENDIX L PLASMIDS USED Table L-1. The plasmids used throughout this dissertation. Plasmid Origin, Insert Marker Backbone Size pJPA105 2 rDNA repeat (XmaI endpoints) TRP1 pAF15 14,829 pJPA106 2 rDNA repeat (AhdI endpoints) TRP1 pAF15 14,746 pJPA107 2 rDNA repeat (PsiI endpoints) TRP1 pAF15 14,747 pJPA113 ARS1 URA3 pRS306 (77) 4,575 pJPA114 rDNA ARS URA3 pRS306 4,575 pJPA116 ARS1 CEN4 URA3 pRS306 5,316 pJPA117 rDNA ARS, CEN4 URA3 pRS306 5,316 pJPA118 rDNA repeat (XmaI endpoints) URA3 pRS306 13,556 pJPA133 ARS1 LEU2 pRS305 (77) 5,698 pJPA136 ARS1 CEN4 LEU2 pRS305 6,468 pJPA138 2 URA3 pRS306 5,703 pJPA148 2 LEU2 pRS305 6,826 pAF15 2 LoxP TRP1 pRS424 (77) 5,692 pAF31 ARS1 URA3,ADE2 pJPA113 6,828 pAF32 ARS1 CEN4 URA3,ADE2 pJPA116 7,568 pRS305 Integrating Vector LEU2 N/A 5,504 pRS306 Integrating Vector URA3 N/A 4,381 pRS424 2 High Copy Vector TRP1 N/A 5,616

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96 APPENDIX M STRAINS USED Table M-1. The strains used throughout this dissertation. Strains Summary Genotype Parent Strain W303R5 WT MAT a leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 N/A W303AR5 ADE2::rDNA MAT a leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 ADE2::rDNA W303R5 yAF5 pRS305 integration MAT a leu2-3,112::pRS305(LEU2) his3-11,15 ura3-1 ade2-1 trp1-1 can1100 RAD5 ADE2::rDNA W303AR5 yAF6 pRS306 integration MAT a leu2-3,112 his3-11,15 ura31::pRS306(URA3) ade2-1 trp1-1 can1100 RAD5 ADE2::rDNA W303AR5 yAF7 cir 0 MAT a leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 ADE2::rDNA cir 0 W303AR5 yAF8 cir 0 MAT a leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 ADE2::rDNA cir 0 W303AR5 W1588-4C WT MAT a leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 W303R5 U952-3B sml1 MAT a sml1::HIS3 leu2-3,112 his311,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 W1588-4C U953-61A mec1 MAT a mec1::TRP1 sml1::HIS3 leu23,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 W1588-4C

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97 can1-100 RAD5 U960-5C rad53 MAT a rad53::HIS3 sml1-1 leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1100 RAD5 W1588-4C yAF2 sml1 MAT a sml1::HIS3 leu2-3,112 his311,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 ADE2::rDNA W303AR5 HKY580-10D WT MAT (alpha) leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 RAD5+ W303R5 RMY178-1A fob1 MAT a fob1::URA3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 RAD5+ HKY580-10D RMY206-5B sir2 MAT (alpha) sir2::HIS3 hmr::TRP1 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 RAD5+ HKY580-10D T2-3D WT Diploid (WT at all markers) N/A GG621 acs1 acs1::APT1 T2-3D GG625 acs2 acs2::Tn5BLE T2-3D yAF10 WT HIS3 MAT a leu2-3,112::pRS305(LEU2 ) ura31 ade2-1 trp1-1 can1-100 RAD5 ADE2::rDNA yAF5 yAF1 yca1 MAT a yca1::HIS3 leu2-3,112 his311,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 ADE2::rDNA W303AR5 W2889-19B shu1 MAT a shu1::HIS3 leu2-3,112 his311,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 W1588-4C U1672 shu2 MAT (alpha) shu2::K. lactis URA3 leu23,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 W1588-4C W4154-3D shu3 MAT a shu3::KANMX leu2-3,112 his311,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 W1588-4C

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98 11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 W3824-21D csm2 MAT a csm2::KANMX leu2-3,112 his311,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 W1588-4C W4173-16B shu1, shu2, shu3. csm2 MAT a shu1::HIS3 shu2::K. lactis URA3 shu3::KANMX csm2::KANMX leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 W1588-4C

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105 BIOGRAPHICAL SKETCH I was born in San Jose, CA on January 7, 1979. At the age of five I moved to Ormond Beach, FL. I went Seabreeze Senior High School (in Daytona Beach, FL) where I graduate as the valedictorian. In August 1997, I started at the University of Florida (UF). In August 1999, I received a Bachelor of Science in Biochemistry from UF. That same month I started the Interdisciplinary Program in Biomedical Sciences at UF, to work on my Ph.D.