Ziv Itzkovich MSC Thesis

TEL-AVIV UNIVERSITY
GEORGE S. WISE FACULTY OF LIFE SCIENCES GRADUATE SCHOOL

School of Molecular Cell Biology & Biotechnology
Regulatory roles of the Elg1 PCNA-unloader in gene silencing and

sister chromatid cohesion in Saccharomyces cerevisiae
Thesis submitted for the M.Sc. degree in Genetics at Tel-Aviv University
By
Ziv Itzkovich
200834505
Under the supervision of
Prof. Martin Kupiec
February 2022
1
Table of contents
1. Abstract ........................................................................................................................................... 3
1.1 CHAPTER 1: ....................................................................................................................................... 3
1.2 CHAPTER 2: ....................................................................................................................................... 4
2. Introduction ........................................................................................................................................ 5
2.1. Genome maintenance.................................................................................................................. 5
2.2. The DNA clamp PCNA................................................................................................................... 6
2.3. The PCNA unloader Elg1 .............................................................................................................. 7
2.4. Chromatin remodeling and Silencing control .............................................................................. 9
2.5. Cohesin and Sister Chromatid Cohesion .................................................................................... 11
3. Materials and Methods ..................................................................................................................... 13
3.1. Growth media ............................................................................................................................ 13
3.2. Yeast Strains ............................................................................................................................... 13
3.2. List of plasmids........................................................................................................................... 16
3.3. Molecular biology techniques .................................................................................................... 18
3.4. Yeast genetics ............................................................................................................................ 22
4. Results ............................................................................................................................................... 23
4.1 CHAPTER 1: ..................................................................................................................................... 23
4.1.1 The role of ELG1-mediated PCNA unloading in heterochromatin formation.......................... 23
4.1.2 Testing the functionality of Elg1 through various mutations .................................................. 26
4.1.3 Testing Elg1 mutants for DNA damage response .................................................................... 31
4.1.4 PCNA levels on the chromatin correlate with heterochromatin and DNA damage sensitivity
phenotypes ....................................................................................................................................... 33
4.2 CHAPTER 2: ..................................................................................................................................... 37
4.2.1 Deletion of Elg1 increases cell viability in the absence of cohesion subunit Pds5 .................. 37
4.2.2 Δpds5 Δcln2 suppression is enhanced by PCNA accumulation on chromatin in elg1 mutants
.......................................................................................................................................................... 41
4.2.3 ∆elg1’s contribution to rescue the SCC phenotype of pds5 is a possible result of Srs2
recruitment to the chromatin by SUMOylated PCNA....................................................................... 43
5. Discussion ......................................................................................................................................... 46
5.1 CHAPTER 1: ..................................................................................................................................... 46
Elg1-mediated PCNA unloading as a driving force behind Δelg1 silencing and DNA repair
phenotypes ....................................................................................................................................... 46
5.2 CHAPTER 2: ..................................................................................................................................... 49
Deletion of Elg1 promotes SCC through SUMO-PCNA accumulation & Srs2 recruitment ............... 49
6. References ........................................................................................................................................ 51
2
1. Abstract
1.1 CHAPTER 1:
In Saccharomyces cerevisiae, the sliding DNA clamp PCNA plays a central role in DNA
replication and repair. PCNA, present on the leading strand and on each of the Okazaki
lagging strands, acts as a landing platform to increase the processivity of different DNA
polymerases, as well as a signaling hub for other proteins. PCNA is loaded onto the DNA by
the RFC complex and is unloaded from the DNA by the RFC – like complex that contains the
subunit Elg1.
Different regions of the genome can be in an open, accessible state (euchromatin) or in an

inaccessible state that precludes their expression (heterochromatin). Post-translational
modifications of histones define these states. During DNA replication, nucleosomes are
evicted in front of the moving fork. The inheritance of epigenetic traits (epigenetic memory)
requires that modified histones should be re-positioned onto the two newly replicated DNA
molecules. Despite this massive reorganization of the chromatin structure, silencing of
transcription is maintained throughout S-phase.
Mutations in the PCNA-unloader Elg1 confer various phenotypes, including sensitivity to

DNA damaging agents, sister-chromatid cohesion defects and hyper-recombination. They
also bring about significant silencing loss at the usually silent HML and HMR loci. Our goal
was to define the mechanism by which Elg1 affects epigenetic memory. We tested a series
of Elg1 alleles containing different site-specific mutations that interfere to different degrees
with its PCNA unloading abilities for their ability to silence the HMR locus of yeast. We found
that the degree of effect in this assay, similar to the sensitivity to DNA damage, correlated
with the amount of PCNA left on chromatin in the different mutants. These results
demonstrate the overlap and coordination between DNA replication, repair and chromatin
remodeling, and the multi-systemic roles of the DNA clamp PCNA, as well as the many
responsibilities of its regulator the unloader Elg1.
3
1.2 CHAPTER 2:
During DNA replication, the sister chromatids are held together until anaphase when they
will separate into the opposite sides the dividing cell. Proper execution of the process
termed Sister Chromatid Cohesion (SCC) is vital for correct segregation of sister chromatids,
the folding of chromosomes into loops, and gene expression. The main protein behind SCC
is the cohesin complex, which holds the sister chromatids together from S phase.
Cohesin is a massive complex comprised of 2 elongated proteins called Smc1 and Smc3,
that are bridged by the kleisin subunit Mcd1/Scc1. A third layer of cohesin subunits involves
Scc3/Irr1, Wpl1/Rad61, and Pds5, which interact with cohesin through its kleisin subunit.
The HAWK subunit Pds5 is essential for cohesion, but its role is not yet fully understood.
Deletion of the PCNA unloader Elg1 can suppress the lethality of a temperature sensitive
allele of Pds5, but not a full deletion of the protein.
Our lab carried out two independent 5-FOA based screens for spontaneously arising
suppressors of Δelg1Δpds5. Our results have shown that cells can stay alive in the absence
of Pds5 on two conditions: one is elevation in the level of Mcd1 (which can be through
mutation in the G1 cyclin Cln2) and the second is elevation in the level of SUMO-modified
PCNA on the DNA (caused by the lack of PCNA loading by Elg1). The elevated level of SUMO-
PCNA increases the recruitment of the helicase Srs2, which works to evict Rad51 molecules
from the moving replication fork. This in turn releases increased amounts of single stranded
DNA that serves as sites for increased cohesion loading and improved SCC establishment.
These results demonstrate the stabilizing role of Pds5 in the cohesin complex for successful
SCC, as well as reveal an alternative route through which lack of Elg1 activity contributes to
overcoming the absence of Pds5.
4
2. Introduction
2.1. Genome maintenance
The stability of the genome is a basic requirement for proper functioning and survivability of
all organisms. Genome stability exists under the constant threat of external forces such as
DNA damaging agents(1), and by internal forces such as the normal metabolism of the cell
(2). To make matters even more complicated, during DNA replication, the unwinding of the
double helix will expose the DNA to different alterations and chemical modifications(3).
So, the challenges cells face to maintain the integrity of their genomes are vast to say the
least. In order to rise to these challenges, eukaryotic cells have developed faithful programs
for DNA replication, repair and chromosome segregation as well as checkpoint signaling
pathways for cell cycle control. Successful genome maintenance requires for all of them
work with extreme precision, perfect coordination, and great speed(4, 5).
The execution of these programs requires, apart from the replicative polymerases, many
other actors that are needed at different locations and different times of the cell cycle.
Histone chaperons act to unravel the chromatin in front of the advancing replication fork, as
well as to seal it again behind it(6, 7), PCNA modifying enzymes work to recruit dedicated
polymerases in the face of DNA damage(8), and cohesin complexes needed for chromosome
segregations are placed around the DNA already before S-phase(9).
The way through which cells orchestrate these multiple activities is not yet fully understood,
but model organisms like Saccharomyces cerevisiae have provided us with genomic tools
such as the systematic creation of mutants in all nonessential and essential gene. Through
such whole-genome approaches it is possible to identify genes with roles in genome
maintenance(10–12).
While a small amount of genome instability is the driver of evolutionary change and is
allowed, defects in genome maintenance almost always results in reduced fitness or cell
death(13). Genome instability is a known characteristic of cancer development(14), which
have raised the need to understand the mechanisms behind genome maintenance.
5
2.2. The DNA clamp PCNA
PCNA (Proliferating Cell Nuclear Antigen) is a homotrimer ring molecule that surrounds the
DNA and acts as a processivity factor for the polymerase enzymes that are required for DNA
replication. DNA replication is an asymmetrical process by nature: The leading strand of the
DNA is copied continuously by polymerase Epsilon (15) , which moves with the fork, and
requires, in principle, a single PCNA molecule; the lagging strand of the DNA moves in the
opposite direction and is copied in a non-continuous manner by polymerase Delta(16) ,
resulting in numerous DNA pieces that undergo later ligation and thus requires many PCNA
molecules.
DNA replication presents many challenges that can be divided into 3 categories: 1 -
steadfast replication of the complete genome (including situations in which there is DNA
damage), 2 - faithful replication of the chromatin structure, and 3 - the establishment of
sister chromatid cohesion. PCNA has been shown to be an important actor in all the above
(17–19); it does so through its many interactions with the various participating proteins.
Such proteins usually hold within them specific motifs designated for interaction with PCNA
(20). In the presence of DNA damage, PCNA turns into a signaling platform, as it can undergo
different modifications that will signal the different proteins at work to undertake certain
actions in response. The modification of PCNA by the addition of a Ubiquitin molecule at
lysine 64 (by the Rad6-Rad18 E2-E3 complex) will signal for activation of the DNA damage
tolerance pathway (DDT). If, during DNA replication, the replisome stalls at a lesion (such as
a deformed nucleotide, a bound peptide or a secondary structure), special DNA polymerases
are recruited that are able to bypass DNA lesion by replicating damaged DNA at the expense
of accuracy. These enzymes are called Translesion synthesis (TLS) polymerases (21).
Alternatively, if the ubiquitin at lysine 64 is further poly-ubiquitinated by Ubc3-Mms2 (E2)
and Rad5 (E3), it activates a different, error-free pathway known as template switching (TS),
whose details are still unclear but involves copying the missing data from the sister
strand(22). A third option is for PCNA to go through modification of a Ubiquitin-like molecule
called SUMO at K64 or K127 by Ubc9 (E2) and the SUMO ligase Siz1(21). These modifications
have been shown to negatively regulate an additional pathway in which an ATP dependent
3’ to 5’ helicase called Srs2 is recruited to SUMO-PCNA, to prevent homologous
recombination(23).
6
PCNA regulation is attributed to a group of protein complexes called clamp loaders and
unloaders, which together control PCNA turnover on the chromatin. The best characterized
among these is Replication factor C (RFC), the canonical loader of PCNA. The RFC complex
has 5 subunits (called Rfc1-5 (19)) that belong to the ATPase protein family that has specific
AAA+ domains, as they use ATP to open PCNA prior to loading it onto the DNA (24). RFC can
as well unload PCNA from the DNA, but it is believed to be essential for loading only(25). In
addition to RFC, three RFC-like complexes (RLCs) exist, in which Rfc1 is replaced by either
Elg1, Ctf18 or Rad24 (26).
2.3. The PCNA unloader Elg1
While the action of PCNA loading onto the DNA by Rfc1-RFC is critical, the action of its
unloading is almost equally critical. The timely unloading of PCNA as well as the control of
PCNA molecules on the DNA is crucial for many cellular properties as discussed above, as
well as for overall genomic stability and the successful advancement of the cell cycle (27, 28).
The main protein complex responsible for unloading PCNA from the chromatin is Elg1-RLC.
In this complex the protein Elg1 replaces Rfc1 in interacting with the Rf2-5 subunits, thus
creating an RLC (RFC–like) complex(29). Elg1 stands for Enhanced Levels of Genomic
instability, as the protein was discovered several times in genome-wide screens that
searched for mutants with phenotypes related to genome instability (30). When a yeast cell
is deleted for Elg1 it is still viable, but it shows many different phenotypes. The phenotypes
include increased rates of gross chromosomal rearrangements and chromosomes loss (30,
31), elevated levels of Ty transposition(32) increased levels of homologous recombination
(28, 33) as well as elongated telomeres(34, 35), high amounts of spontaneous DNA
damage(36, 37), and increased sensitivity to various DNA damaging agents.(38, 39). Mutants
lacking Elg1 also exhibit defects in sister chromatid cohesion (SCC) and are synthetic lethal
with hypomorphic alleles of cohesin subunits(40). When expressed at high amounts Elg1 can
positively regulate the DNA repair process known as “salvage recombination”, in which the
information for DNA synthesis is copied from the sister chromatid, a pathway that is
repressed by the recruitment of the Srs2 helicase(23). As mentioned, in order to operate as
an unloader of PCNA, Elg1 creates the Elg1-RLC complex through interactions with RFC
7
subunits Rfc2-5. Interestingly, Elg1 expression level is 3-fold lower than that of the other
subunits (41) . Elg1’s structure includes a central domain with a conserved ATPase region,
flanked by unique N-terminal and C-terminal region (42). Given that PCNA loading is an ATP
based process, all PCNA loaders have an ATPase domain (AAA+) that contains conserved
motifs that participate in ATP binding and hydrolysis called Walker A and Walker B motifs,
respectively (43, 44). Elg1 carries in its AAA+ domain regions with homology to Walker A and
B domains, although they deviate from the consensus sequence (45) and Elg1’s AAA+
domain is not believed to be catalytically active (36) even though the overall complex Elg1-
RLC may still carry ATPase activity through the AAA+ domains of the Rfc2-5 subunits. In its
N-terminus region, Elg1 has motifs capable of carrying out non-covalent interactions with
SUMO-PCNA. These motifs are termed SIM for SUMO Interacting Motif (46). SIMs are
characterized by a series of hydrophobic amino acids (usually Valine and Isoleucine) that are
followed by several amino acids of negative charge. (47, 48). Elg1 also holds in its N-terminus
region (from AA 43 to 67) a motif that fits the consensus PIP motif (PCNA Interacting
Peptide) for direct PCNA binding. PIPs are a known component of different PCNA related
proteins, and their structure has been documented for a substantial number of proteins
with a canonical PIP box derived (20, 49). When tested for their ability to sensitize cells to
DNA damage, mutations in the SIM and PIP motifs have not shown to hinder cell growth on
the DNA damaging agent MMS, hinting that, Elg1 and PCNA hold more interaction sites able
to compensate for their absence (50). In vitro assays have shown how treating chromatin
with purified ELG1-RLC resulted in PCNA unloading(51), and it has been shown in vivo that
Elg1 unloading activity necessitates ligation of the newly synthesized DNA strand(51). Elg1
accumulates at the lagging strand, and deletion of Elg1 leads instead to accumulation of
PCNA on the lagging strand(52). All this supports the notion that Elg1 is the main unloader of
PCNA.
8
2.4. Chromatin remodeling and Silencing control
DNA in all eukaryotes is tightly pack in an extremely effective manner. This unique 3D
structure holds within it the ability to control gene expression and overall phenotypes. The
structure is made up of several layers of complexity, beginning with the wrapping of the
DNA around nucleosomes, eight-part complexes consisting of pairs of histones, H2A, H2B,
H3 and H4 (53). The clustering of these structures along the chromatin determines the
availability of DNA for gene expression. DNA in heterochromatic areas where the
nucleosomes are tightly packed are less accessible for proteins related to transcription to
interact with. In such areas gene expression will be greatly reduced and possibly silenced. In
euchromatic areas nucleosomes are less abundant and more spaced out, making the DNA
more available for normal gene expression. Another aspect that is known to have an effect
is the modifications of histones, which affect the level as well as timing of gene expression,
and are possible to pass through generations in what is known as epigenetic inheritance
(54). S. cerevisiae is known to have two main histone post-translational modifications that
affect gene expression, acetylation and methylation of histone H3 or H4 (55, 56). Histone
acetyltransferases acetylates histones to promote euchromatin formation while histone
deacetylases promote heterochromatin formation (57, 58). The reason behind this is the
change in electric charge of a histone upon the addition of an acetyl group, that will remove
the overall positive charge of the histone, which will in turn decrease its attraction to other
histones as well as to the DNA itself, all of which will result in a more ‘open’ DNA (7). The
primary force behind heterochromatin creation in yeast cells is the Sir (Silent information
regulator) complex. For silencing to be established, Sir needs to be recruited to special
regulatory ‘’Silencer” sites that flank the area to be silenced (59–61). This recruitment is
mediated through the activity of various proteins including Abf1, Rap1, ORC (origin-
recognition complex) proteins as well as DNA silencer proteins (62). The Sir complex itself is
comprised of 3 subunit proteins named Sir2, Sir3 and Sir4. Sir2 is the catalytic subunit of the
complex, and functions as a deacetylase and removes acetyl modifications from H3 and
H4(63). When Sir2 is paired with the other subunits, the complex binds proteins with low
acetylation levels and facilitates their spread throughout the chromatin (64). How do these
intricate structures behave during DNA replication? As nucleosomes are separated from the
DNA by the advancing replisome, they move behind the replication fork, to be distributed to
9
the new DNA at random(6). The accurate copying of histone modifications from the
template DNA is mediated by a group of histone chaperons such as CAF1, Rtt106, Asf1 and
the FACT complex, which work in coordination with the components of the replisome (65).
Asf1 has been shown to interact with the RFC subunit Rfc1 (66) while Rtt106 has been
shown to interact with the RLC subunit Elg1 (67) both of which help explain their
recruitment to the replication fork. Asf1 and Rtt106 are also in charge of acetylating lysine
56 of histone H3 (H3K56Ac). Since behind the fork there is twice the amount of DNA, new
histones are deposited together with the transferred ones. These new histones are marked
by the H3K56Ac mark (68). Following interaction with Asf1, histones are transferred to CAF1,
a chromatin remodeler for deposition onto the DNA to create new nucleosomes (69).
Coordination between CAF1 and the replisome happens through PCNA, which recruits CAF1
through interaction with its Caf1 subunit (70, 71). After the passing of the replication fork
and the reassembly of nucleosomes, heterochromatin will be established by the Sir
complex.
10
2.5. Cohesin and Sister Chromatid Cohesion
After DNA replication the 2 chromatids of DNA are held together at close vicinity by a
protein complex called Cohesin. This phenomenon is called Sister Chromatid Cohesion
(hereafter SCC) and it lasts until the end of anaphase, after which the two chromatids will
separate to opposite sides (72, 73) by the force exerted by the spindle microtubule
apparatus (74, 75). SCC is an extremely important process during a cell’s life, and defects in
the loading of cohesin on to the DNA, establishment and maintenance of cohesion or the
cleavage of cohesin for DNA detachment have all been shown to lead to chromosome loss
and cell death (76)
Cohesin is made up of two coiled rod-shaped proteins called Smc1 and Smc3 (Structural
maintenance of the Chromosome) that together form a V-shaped heterodimer. They hold
within them distinct terminal domains called ATPase head and Hinge that are connected to
each other (77, 78). Additional proteins bind to the heterodimer including the Kleisin subunit
Scc1/Mcd1 (hereafter Mcd1) that interacts with Smc3’s head domain through its N-terminus
and Smc1 through its C-terminus (77–80). Mcd1’s interaction with cohesin as well as
cohesin’s association with the chromosome are ATP dependent(81, 82). Another protein
called Scc3 binds cohesion through Mcd1, while accessory interchanging subunits including
Pds5, Scc2, Scc4, Wpl1 and Eco1, are needed for cohesion regulation(73). Scc2 and Scc4
form a complex that loads cohesion on the chromatin at the kinetochores and along the
chromosomes during late G1 and S phases(83, 84) .Eco1 is an essential acetyltransferase that
acetylates cohesion components during S phase (85, 86) it acetylates Smc3 at K112 and
K113, stabilizing it against Wpl1-dependent destabilization (87). Eco1 is recruited to the
chromatin by PCNA, and it holds within it a PIP motif for direct PCNA interaction (88). Also,
Smc3 acetylation has been shown to peak during S-phase and is a cell cycle regulated
process (89). Put together, these last two facts point to cohesion establishment being
another replication-coupled process.
The HAWK subunit Pds5 is important for two seemingly opposing roles, as it is needed for
the complex’s maintenance and for promoting the acetylation of Smc3 by Eco1 (90, 91) on
one hand, but together with Wpl1 it is also needed for destabilizing cohesin to detach it
11
from the chromatin (92). Cohesion is maintained until anaphase when the separase Esp1
cleaves Mcd1, breaking cohesion’s hold on the sister chromatids (93).
Elg1 is likely also playing a role in SCC, as it is known to interact with SCC components (26).
When Elg1 deletion is paired with deletion of Ctf4, a replisome protein that recruits
polymerase Alpha, the helicase Chl1 as well as other proteins(94, 95), cells are rendered
inviable, and overexpression of Mcd1 or Scc2 rescues this phenotype. Deleting Elg1 on the
background of temperature sensitive alleles of Smc3 or Mcd1 further reduces their fitness.
However, when Elg1 is deleted on the background of a temperature sensitive alleles of Pds5
and Eco1 cells are viable again(96, 97). All these results hint at a role for Elg1 in SCC
regulation.
12
3. Materials and Methods
3.1. Growth media
YEAST:
YPD (yeast rich medium) - 1% Bacto yeast extract, 2% Bacto peptone, 2% Glucose.
SD (yeast defined medium) - 0.67% Bacto yeast nitrogen base w/o amino acids, 2% Glucose. Amino
acids were added according to requirement.
SM - 1% potassium acetate (KAC).
BACTERIA:
LB - 1% Bacto Tryptone, 0.5% Bacto yeast extract, 0.17M NaCl. Ampicilin 50 mg/l was added
to LB+Amp plates.
3.2. Yeast Strains
Table 1: Yeast strains – CRASH strains, PCNA disassembly prone mutants
Mating
Number Genotype Source
type
MAT@, ADE2 lys2 TRP1 hmlα2Δ::CRE Kupiec
16770 @
ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tADH1 lab
MAT, @ ADE2 lys2 TRP1 hmlα2Δ::CRE
Kupiec
16904 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tADH1 elg1::ura3
lab
(102)
MAT@, ADE2 lys2 TRP1 hmlα2Δ::CRE
This
19802 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD,pol30-
study
S152P::LEU2.
This
study
V180D::LEU2.
13
This
study
D150E::LEU2.
This
19858 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD,elg1::ura3,pol30-
study
D150E::LEU2.
This
study
E143K::LEU2.
This
study
S152P::LEU2.
This
19890 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD,elg1::ura3
study
,pol30-V180D::LEU2.
This
19950 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD , pol30-
study
E143K::LEU2.
Table 2: Yeast strains – CRASH strains, Elg1 mutants
Mating
type
16770 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD, elg1 SN This Study
57,58 AA -13myc::KanMX
19401 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD, elg1 I This Study
28,93,121,122 AKAA TT386/7DD-13MYC ::KanMX
14
19403 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD, elg1 This Study
KK343/4DD-13MYC::KanMX
∆aa 282-319 replaced with linker of 5aa::GCACG -13MYC::KanMX
TT386/7AA-13myc::KanMX
28,93,121,122 AKAA TT386/7AA-13myc ::KanMX
28,93,121,122 AKAA-13 myc::KanMX
V390A-13myc::KanMX
KK343/4AA-9myc::KanMX
TT386/7DD-13myc::KanMX
19425 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD, This Study
Elg1D407A,D409A -13myc::KANMX
Elg1(D407K, D409K)WALKER b -13myc::KANMX
15
19428 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD, Elg1 This Study
KK343/4DD+ D407A, D409A -13myc::KANMX
ELG1::13MYC::KANMX
20491 @ ura3Δ::pGPD:loxP:yEmRFP;tCYC1:hygMX:loxP:yEGFP:tAD, Elg1- This Study
V390D::13MYC::KANMX
E134 mat @ ade5-1,lys2::InsEa14,trp1-289,his7-2,leu2-
19369 @ Kupiec lab
3,112,ura3-52,
E134 mat @ ade5-1,lys2::InsEa14,trp1-289,his7-2,leu2-
20436 @ Kupiec lab
3,112,ura3-52, elg1::Hyg,
3.2. List of plasmids
Table 1: Elg1 mutants

pRS415 elg1 SN 57,58 AA -13myc::KanMX Centromeric expression
4327 Kupiec lab
vector
pRS 415 elg1 I 28,93,121,122 AKAA TT386/7DD-13MYC ::KanMX

4328 Kupiec lab
Centromeric expression vector
pRS415 elg1 KK343/4DD-13MYC::KanMX Centromeric expression

4329 Kupiec lab
vector
pRS415 elg1 ∆aa 282-319 replaced with linker of 5aa::GCACG -

4330 Kupiec lab
13MYC::KanMX Centromeric expression vector
pRS415 elg1 TT386/7AA-13myc::KanMX Centromeric expression

4331 Kupiec lab
vector
16
pRS415 elg1 I 28,93,121,122 AKAA TT386/7AA-13myc ::KanMX
4332 Kupiec lab
pRS415 elg1 I 28,93,121,122 AKAA-13 myc::KanMX Centromeric

4333 Kupiec lab
expression vector
4334 pRS415 elg1 V390A-13myc::KanMX Centromeric expression vector Kupiec lab
pRS415 elg1 KK343/4AA-9myc::KanMX Centromeric expression

4335 Kupiec lab
vector
pRS415 elg1 TT386/7DD-13myc::KanMX Centromeric expression

4336 Kupiec lab
vector
pRS415 elg1D407A,D409A -13myc::KANMX Centromeric

4337 Kupiec lab
expression vector
pRS415 elg1(D407K, D409K) WALKER b -13myc::KANMX
4338 Kupiec lab
pRS415 elg1 KK343/4DD+ D407A, D409A -13myc::KANMX
4339 Kupiec lab
Prs415 elg1-V390D::13MYC::KANMX Centromeric expression
4340 Kupiec lab
vector
4190 pRS415 empty vector Centromeric expression vector Kupiec lab
4343 Prs415 ELG1::13MYC::KANMX Centromeric expression vector This Study
pRS425 elg1 I 28,93,121,122 AKAA High copy number expression
4356 This Study
vector
pRS425 elg1 KK343/4DD + DD407,409AA High copy number
4357 This Study
expression vector
4358 pRS425 elg1 DD407,409KK High copy number expression vector This Study
4359 pRS425 elg1 DD407,409AA High copy number expression vector This Study
pRS425 elg1 I 28,93,121,122 AKAA TT386/7DD High copy number
4360 This Study
expression vector
pRS425 elg1 I 28,93,121,122 AKAA TT386/7AA High copy number
4361 This Study
expression vector
17
3.3. Molecular biology techniques
E. coli Plasmid extraction
Plasmids were extracted from E. coli using the DNA-spin plasmid DNA purification kit
(Macherey-Nagel).
E. coli transformation
Competent cells were thawed on ice. 50-100 µl bacteria were mixed with 1µl DNA, and
incubated at 42ºC for 2 min. Following heat shock cells were immediately transferred to ice
for 2 min and afterward resuspended in 1ml LB and were allowed to grow at 37º for 1hr.
Appropriate dilutions were plated on selective plates (LB+ Amp) and allowed to grow over
night at 37ºC.
Yeast chemical transformation
Yeast overnight cultures were diluted 1/20 into 50ml of YPD and incubated at 30ºC, 200
RPM for 3-4 hours. Cells were harvested at a density of 4-8*107 cells/ml, washed twice with
H2O and resuspended at a final volume of Autoclaved DDW. 100µl of the cell suspension
was then added to 360µl transformation mix (33% PEG3500, 0.1M Lithium Acetate, 0.27
mg/ml Salmon Sperm Single Stranded DNA), 200 ng-1µg of plasmid or fragment DNA was
added and the mixture was vortexed gently and incubated at 30ºC for 20-40 min, and then
at 42ºC for 30 min. Following heat shock cells were spun down, resuspended in 200μl of
DDW, and then plated on selective plates.
Chromatin fractionation assay
Cells from 50 ml cultures (OD600<1.0) were collected by centrifugation, successively

washed with ddH2O, PSB (20mM Tris–Cl pH 7.4, 2 mM EDTA, 100 mM NaCl, 10 mMb-ME)
and SB (1 M Sorbitol, 20 mM Tris–Cl pH 7.4), and transferred to a 2 ml Eppendorf tube. Cells
were suspended in1mlSB,30l Zymolase 20T (20 mg/ml in SB) was added, and samples were
incubated at 30◦C with rotation until>85% spheroplasts were observed (60–90 min).
18
Spheroplasts were collected by centrifugation (2K, 5 min, 4◦C), washed twice with SB, and
suspended in 500 ml EBX (20mM Tris–Cl pH 7.4, 100 mM NaCl, 0.25% Triton X-100,15 mM-
ME + protease/phosphatase inhibitors). TritonX-100 was added to a 0.5% final concentration
to lyse the outer cell membrane, and the samples kept on ice for 10 min with gentle mixing.
The lysate was layered over 1 ml NIB (20 mM Tris–Cl pH 7.4, 100 mM NaCl, 1.2 M sucrose,
15 mM-ME + protease/phosphatase inhibitors) and centrifuged at 12K RPM for 15 min, at
4◦C. The super-natant (cytoplasm) was discarded. The glassy white nuclear pellet was
suspended in 500l EBX and Triton X-100 was added toa 1% final concentration to lyse the
nuclear membrane. The chromatin and nuclear debris were collected by centrifugation (15K,
10 min, 4◦C). Chromatin was suspended in 50l Tris pH 8.0 for western blot analysis
(Chromatin). To each fraction an equal volume of 2×SDS-PAGE loading buffer (60 mM Tris
pH 6.8, 2% SDS, 10% glycerol, 0.2%bromophenol blue, 200 mM DTT) was added, samples
were incubated at 95◦C for 5 min and were then analyzed by SDS-PAGE and western blot
analyses.
Protein Western Blot
Alkaline lysis: Cells were grown on standard medium to a concentration of 2x10 7 cells/ml.
Cells (1ml) were washed with DDW, resuspended in 100 μl solution containing 20 μM 5%
NaOH, 0.5% Beta-Mercapto-Ethanol and incubated for 30 minutes on ice. Then 1.6 μl Hcl
and 25 μl sample buffer were added to the tube and boiled for 3 minutes.
SDS-PAGE:
Resolving gel: 30% Acrylamide, 1.5 M Tris-HCl pH8.8, 10% SDS (pH 7.2), 9.7 ml H2O, 100 µl
10% APS and 10 µ TEMED.
Stacking gel: 30% Acrylamide, 1 M Tris-HCl pH 6.8, 10% SDS (pH 7.2), 5.5 ml H2O, 800 µl 10%
APS and 8 µl TEMED.
The samples were run with SDS-PAGE Buffer at 100V until the samples have passed the
stacking gel and then at 160V till the samples have been fully separated. Transfer to
nitrocellulose was done in transfer buffer (200 ml Methanol, 3.03gr Tris Base, 14.4 gr
Glycine) at 500mAMP and verified by staining with Ponceau-S dye. The blot was blocked
with Milk for at least 60 minutes at RT. Primary antibody was added for 12 hours at 4°C. The
19
blot was washed 3 x 5 minutes with TBST (Tris-Buffered Saline Tween-20) and secondary
antibody was added for 1 hour. The blot was washed 3 x 5 minutes with TBST and subjected
to ECL.
Yeast genomic DNA extraction
Phenol method - 5ml of a stationary yeast culture was harvested, and the supernatant was
removed. To the tube we added ~0.3g acid-washed glass beads, 500µl lysis buffer (2%
Triton, 1% SDS, 0.1M NaCl, 10mM TrisHCl pH 8, 1mM EDTA) and 300µl 25:24:1
phenol:chloroform:isoamyl alcohol. Tubes were vortexed for 30 min and centrifuged for 15
minutes at 14,000 rpm. 400µl of the top phase was carefully transferred into a new tube
containing 1ml of ethanol 100% to precipitate the DNA. Tubes were mixed and then
incubated for 10 min at RT and centrifuged for 20 min at 14,000 rpm at 4ºC. The pellet was
resuspended in 300µl TE x1 (10mM TrisHCl pH 7.5, 1mM EDTA) with RNase (Sigma) 50µg/ml
in 65ºC for 10 min. DNA was then precipitated by adding 110µl of 10M Ammonium acetate
and 1000µl of ethanol 100% and incubated at -70ºC for 2-16 hrs. Tubes were centrifuged for
20 min at 14,000 rpm at 4ºC. The DNA pellet was washed with cold 70% ethanol and
resuspended in in 50µl H2O.
FACS
Cells were grown overnight, and then diluted again in fresh medium, for growth to mid-log
in the indicated temperature. Cells were then synchronized with α-factor (40ng/µl for bar1
cells) for 2 hours, and synchronization was assessed by visual inspection. Cells were then
incubated with auxin (5mM) for 1.5 hours, then washed 5 times with fresh medium
containing auxin before incubating in shaking conditions. Samples were taken every 20
minutes, centrifuged and washed with 200µl of TE solution (50mM TRIS pH=7.5, 10mM
EDTA). Cells were then pelleted and resuspended in 140µl of ice-cold ethanol 100% and 60µl
of TE, and incubated overnight at 4°C. The following day cells were arranged in a 96 wells
plate, centrifuged at 4000RPM for one minute, and the supernatant was disposed of. Cells
were resuspended in 200µl of TE with 0.5mg/ml of RNaseA, and plate was incubated
overnight at 37 degrees. Cells were again centrifuged at 4000RPM for one minute, and
supernatant was disposed of. Cells were resuspended in TE with 0.5mg/ml of Proteinase K
20
solution and incubated for 4 hours at 37 degrees. Cells were again centrifuged at 4000RPM
for one minute, supernatant was disposed of, and the pellet was resuspended in TE solution
with 20µg/ml of Propidium Iodide. Cells were then incubated overnight at 4°C in the dark.
The following day, single cells were de-clumped using three rounds of sonication at 20% (or
20 Joul for 5 seconds), then reads were taken using MACSQuant® flow cytometer machine.
Quantification of Silencing Loss by Flow Cytometry
For each CRASH strain, 10 single colonies were inoculated separately into 2 mL of SD-Trp-
Leu medium in 96-well deep-well plates (VWR) and were grown overnight to saturation at
30 °C on a microplate orbital shaker (VWR). Overnight cultures were diluted in 1 mL of fresh
medium at a density of 105 cells/mL in 96-well deep-well plates and were grown at 30 °C on
a microplate orbital shaker until midlog phase. For each culture, a minimum of 50,000
events were collected using an Attune NxT Flow Cytometer (Life Technologies). Scatterplots
of forward-scatter (height) and forward-scatter (width) measurements were generated, and
gating was established to include only unbudded and budded cells and to exclude debris
and clumped cells for further analysis. Gating was used to measure separately the number
of GFP+ cells and the number of RFP+ cells. Finally, a Boolean logic gate, RFP+ AND GFP+,
was used to determine the number of cells that were both GFP- and RFP-fluorescent. Such
cells were inferred to have very recently undergone the Cre-mediated recombination event
leading to GFP expression but retained RFP expressed in the recent past. The number of
cells in a population that had very recently lost silencing (cells that were both GFP- and RFP-
fluorescent) was divided by the number of cells in the population that had the potential to
lose silencing (cells that were only RFP fluorescent plus cells that were both GFP-and RFP-
fluorescent) to obtain an apparent rate of silencing loss. Perdurance of RFP molecules in
cells for two or three generations after switching from expression of RFP to GFP precluded
direct calculation of true rates of silencing loss using this method. However, because the
apparent rates of silencing loss directly reflected true silencing loss rates, these values could
be used to quantitatively compare silencing-loss rates across different genetic backgrounds.
21
2-Dot cohesion assay
Cells were grown overnight, and then diluted again in fresh medium, for growth to mid-log
in 25°C. Cells were then synchronized with α-factor (40ng/µl for bar1 cells) for 2 hours.
Cells were washed 5 times with fresh medium containing nocodazole (15μg/μl). Cells were
incubated at the indicated temperatures in shaking conditions, and 200 cells were classified
according to the number of GFP dots observed under the fluorescent microscope.
3.4. Yeast genetics
Mating
Two haploid colonies were mixed on YPD plate and incubated over night at 30ºC. The
mixture was streaked or replicated to appropriate selective plates and grown at 30ºC, until
colonies were visible.
Sporulation
The selective plate for diploids was replicated to solid sporulation medium at 25 0 C and
incubated for 10-12 days.
22
4. Results
4.1 CHAPTER 1:
4.1.1 The role of ELG1-mediated PCNA unloading in heterochromatin formation
Our system for measuring gene expression silencing is a fluorescence-based assay called
CRASH, or Cre-Reported Altered States of Heterochromatin(98). This system measures gene
expression in the yeast silent mating type locus HML. The system has two genomic
components: the first is a gene encoding the Cre recombinase inserted at HML. The second
component is a sequence of RFP and drug resistance genes, placed between 2 LoxP sites.
These LoxP sites are placed downstream to a GPD promoter and upstream to a
promoterless GFP gene. If silencing at the HML is lost, even transiently, and Cre is
expressed, this will lead to site specific recombination between the LoxP sites, which will
result in a permanent switch in expression, from RFP to GFP (Fig 1A). Such silencing-loss
events are manifested as green cells in flow cytometry and appear at low levels in wildtype
cells. Thus, a transient epigenetic event is converted to a permanent genetic change.
When looking at cells deleted for ELG1, a significant increase in silencing loss can be seen
compared to wildtype cells (Fig 1B). Flow cytometry analysis was used to quantify the rate
of RFP to GFP switching. Cells that have switched in the last generation express both RFP
(expressed in the previous generation) and GFP (newly switched). Thus, the number of both
RFP AND GFP positive cells compared to the total number of cells (cells that were only RFP
fluorescent plus cells that were both GFP-and RFP-fluorescent) gives the apparent rate of
silencing loss. We call this "apparent" because potential perdurance of RFP molecules in
cells for two or three generations after switching from expression of RFP to GFP preclude
direct calculation of true rates of silencing loss using this method. However, this calculation
allows a quantitative comparison between strains of different genetic backgrounds ((98)).
The apparent silencing-loss rate in Δelg1 is larger than that in wildtype cells by one order of
magnitude (Fig 1C, D).
By now, it is well established that deletion of Elg1 leads to accumulation of PCNA on the
chromatin (29) . This increase in PCNA levels is believed to be responsible for most genome
instability phenotypes seen in Δelg1 cells (42). This led us to believe that the increase in
23
silencing loss seen for Δelg1 may also be due to excessive retention of PCNA on the DNA. To
further test this we created a series of CRASH strains containing different PCNA mutations.
These mutations, E143K, S152P, V180D are known to cause PCNA to spontaneously
disassociate from the chromatin due to disruptions in the interactions between PCNA’s
subunits (regardless of Elg1-mediated unloading) (99). These mutants are termed
disassembly-prone PCNA mutants (DPP). All DPP mutants showed an ability to almost fully
rescue the silencing loss seen for Δelg1, lowering the silencing loss levels close to those of
the wildtype (Fig 1C, D).
A B
wt elg1
24
D
Figure 1: The PCNA-unloading activity of Elg1 contributed to silencing HML. (A) Illustration
of the CRASH assay to measure silencing of HML. Loss of silencing at HML leads to Cre
expression and results in Cre-mediated recombination between two LoxP sites, resulting in
removal of the RFP and HygMX genes, repositioning the GFP gene so that it becomes
constitutively expressed. (B) elg1 mutants show high levels of silencing. Comperative
images of wild type and elg1 colonies. Loss-of-silencing events manifest as GFP-expressing
in otherwise RFP-expressing sectors. (C) Representative images of RFP-GFP distribution as
viewed with flow cytometry. from wild-type (16770), Δelg1 mutant (16904) and disassembly
prone PCNA mutant strains containing the CRASH assay cells are divided into populations
with RFP omitting cells appearing close to the X axis and GFP omitting cells appearing close
to the Y axis. (D) The apparent silencing-loss rates of the strains in C were quantified as
described in Materials and Methods. ****, P <0.0001, values were calculated using ANOVA
and Sidak’s test post-hoc analysis.
25
4.1.2 Testing the functionality of Elg1 through various mutations
To further investigate the relationship between Elg1–RLC-mediated PCNA unloading and

silencing control, we went on to test the effect of mutations in Elg1 that were not a full
deletion of the protein. We created a series of mutations in Elg1 throughout the protein (Fig
2):
In the N-terminus domain: A PIP (PCNA Interacting Peptide) consensus sequence can be
found at aa 43-67 (50). Within this region we chose to mutate amino acids SN at position
57,58. They were mutated to alanine (AA).
Point mutations I28A I93K II121/2AA (termed SUMO Interaction Motifs or SIMs) affect the
binding between the N terminal of Elg1 and SUMO and were shown to also affect the
interaction with SUMOylated PCNA (50). The last area mutated in the N terminus was an
unstructured loop region, spanning from aa 290 to aa 319 and containing a hydrophobic
patch. This site is a possible protein - protein interaction site and is conserved throughout all
Elg1 orthologs but is absent in other components of the RFC complex and in the other Rfc1-
like proteins (42). We replaced this loop by a small linker peptide.
In the ATPase domain: we chose to mutate two Threonines at position 386,387. By modeling
Elg1 on the known Rfc1 crystal structure, these two residues are predicted to be in the
interphase with PCNA (100). These residues were mutated to either Aspartic acid (hereby
referred to as elg1-386/7DD) or to Alanines (elg1-386/7AA). The two Lysines at positions
343, 344 lie within the Walker A motif and are conserved between Elg1 and its human
ortholog ATAD5(101). These, together with the main chain NH atoms, are thought to be
crucial for ATP binding (42). We mutated them to Aspartic acids (elg1-343/4DD) or to
Alanines (elg1-343/4AA). We also mutated two Aspartic acids in positions 407,409 that lie
within the Walker B motif, also conserved between Elg1 and the Human ATAD5-RLC(101) .
These residues are thought to be crucial for ATP binding and/or hydrolysis and have been
shown to be crucial for PCNA binding(42) . They were mutated either to Aspartic acid (elg1-
407/409DD) or to Alanine (elg1-407/9AA). Finally, we mutated a novel site at position 390
from Valine to Aspartic acid (elg1-V390D) and to Alanine (elg1-V390A). This site contains
partial homology in Elg1 positions 381-390 (LLDFTTTHYV) with a small patch of Yku80 (aa
450-456 LLDrTTTsgV), and the Valine is conserved throughout Elg1 orthologs (102)
26
The C-terminus of Elg1 mediates oligomerization with Rfc2–5, nuclear import, and
chromatin association. Deletion of AA 521-731 have been shown and is critical for the
function of Elg1, while deletion of AA 731-791 (the last 60 AA) was discovered as non-crucial
(36)
Figure 2: Schematic representation of the Elg1 protein showing domains and mutation
sites. In the N terminus mutation sites (from N to C) SIM1, PIP, SIM2, SIM3 and in the AAA
domain mutation sites LOOP, Walker A, TT386/7, V390, Walker B
27
First, we went on to test how the different mutations affect global protein levels via a
western blot assay (as described in Materials and Methods). Cell cultures were grown in YPD
medium to mid log phase, then harvested and total cellular proteins were extracted from
each culture. The samples were then processed through western blot SDS PAGE gel (Fig 3).
This process was repeated independently 3 times and the results were quantified after
normalizing to the protein levels of the house keeping gene Pgk1 (Fig 3). Out of the 14
mutants the following exhibited relative protein levels significantly lower than wildtype
ELG1: 3xSIM, 3Xsim TT386/7AA, 3Xsim TT386/7DD, KK343/4DD+DVD407/409AVA (Walker
A+B) and DVD407/409AVA (Walker B). All other mutants have maintained protein levels on
par or close to wildtype ELG1 (Fig 3).
28
Figure 3: protein levels for the different Elg1 mutation. Pgk1 was used as a loading control.
The graph represents the Western blot quantification of the relative protein abundance of
the different Elg1 mutants in the cell, compared to the relative level of wildtype ELG1 that is
normalized to 100% (mean +/- standard deviation [SD]; n= 3). *, P = 0.0119 / 0.0291, **, P =
0.0011, ***, P = 0.0001, ****, P <0.0001, values were calculated using ANOVA and Dunnett’s test
post-hoc analysis.
Next, all the Elg1 mutations were tested with our designated silencing assay CRASH yeast
strains and had apparent silencing loss index calculated as described in Figure 1.
Mutations in the PIP and SIM motifs had no effect on silencing levels (Fig 4). This hints that
perhaps mutating these sites is not enough to hinder the interaction as Elg1 might have
further interaction sites with PCNA. Mutating TT386/7 to AA increased silencing loss by a
small amount , while mutating to DD instead had a deletion-like effect (Fig 4). It is possible
that the addition of negative charge from the Aspartic acids brought about a change in
protein structure, functionality, or affinity to PCNA that the non-polar Alanines did not.
Coupling SIM mutations with mutations at the Threonines at 386,387 had an additive effect
for elg1-386/7AA but not for elg1-386/7DD, which already shows the maximal effect (Fig 4).
Deletion of the unstructured loop did not have any effect, as did the change of V390 to
Alanine (Fig 4). Changing V390 to Aspartic Acid did bring a significant increase in silencing
loss (Fig 4). This could be explained similarly to the case of the TT386/7 mutations. Mutating
in the Walker A motif (KK343/4) increased silencing loss when changed to DD but not to AA
(Fig 4). Finally, In the walker B area, changing from D to Lysine as well as to Alanine brought
significant silencing losses (Fig 4).
29
Figure 4: The apparent silencing-loss rates of the different Elg1 mutations. CRASH
containing strains were mutated in the genome with KanMX to replace ELG1 with our set of
different elg1 mutants. All strains were quantified by flow cytometry, as described in
Materials and Methods. ****, P < 0.0001 was calculated using ANOVA and Dunnett’s test
post-hoc analysis.
30
4.1.3 Testing Elg1 mutants for DNA damage response
Methyl methane sulfonate (MMS) is a DNA alkylating agent that is used for many years as a
DNA damaging agent. It is used to test the ability of cells to complete DNA replication (as
alkylated DNA is poorly replicated by DNA polymerases and must be efficiently repaired)(1).
All Elg1 mutants cloned in a shuttle plasmid were introduced into a Δelg1 yeast strain
(20436) of a different genetic background from the CRASH strains shown before. All
plasmids were of the pRS415 background (centromeric with a LEU2 marker) and designed to
mimic genomic expression. As controls, either the wild type ELG1 copy, or an empty vector,
were used. All strains were spotted as 5-fold serial dilutions either in SD –Leucine plates
(control), or on SD complete plates with varying amounts of MMS (Fig 5). Wildtype cells are
able to grow at all MMS concentrations presented while Δelg1 cells did not grow past
0.0075% MMS. Among the different Elg1 mutants, MMS sensitivity was seen for TT386/7
when mutated to aspartic acid but not to alanine, or when paired with the 3xSIM mutations.
Mutation V390 seems to show also increased sensitivity when mutated to Aspartic acid but
not to Alanine. Growth sensitivity was also seen for both Walker mutations, with the Lysines
at 343/4 (Walker A region) showing sensitivity when mutated to Aspartic acid but not to
Alanine, and the Aspartic acids at positions 407/9 (Walker B region) showed increased
sensitivity when mutated to both lysine and alanine.
Apart from the mutations mentioned, no other Elg1 mutation within the group increased
MMS sensitivity. Also, All Elg1 mutants that have shown increased DNA damage sensitivity
have also shown an increased level of silencing loss with the CRASH assay. This strong
correlation further supports the notion that retention of PCNA on the DNA in the absence of
Elg1 or with the presence of a mutated less effective one is the main cause for both
phenotypes.
31
Figure 5: DNA damage sensitivity assay for different ELG1 mutations. Serial five-fold
dilutions of yeast cultures on SD-Leu or SD-Complete with methyl methane sulphonate
(MMS) in the indicated concentration shows different sensitivities of elg1 mutants in strains
19369,2043. Strains were transformed with pRS415 plasmid collection containing the
different ELG1 mutations.
32
4.1.4 PCNA levels on the chromatin correlate with heterochromatin and DNA damage sensitivity
phenotypes
Some of the elg1 mutants tested showed decreased stability levels (Figure 3). We were
interested in measuring the PCNA levels on the chromatin of all mutants, but before doing
that we attempted to express PCNA to similar levels in all mutants. We introduced into
Δelg1 cells high copy number plasmids carrying the 3xSIM, 3Xsim TT386/7DD, 3Xsim
TT386/7AA, KK343/4DD+DVD407/409AVA (Walker A+B), DVD407/409AVA (Walker B) and,
DVD407/409KVK (Walker B) alleles (all cloned into pRS425 LEU2 plasmids containing a 2-
micron origin of replication sequence designed for high copy expression). Cell cultures were
grown in -LEU media to mid log phase, then harvested at equal concentrations and total
cellular proteins were extracted from each culture. The samples were then submitted to
western blot analysis (Fig 6). This process was repeated independently 3 times and the
results were quantified after normalizing to the protein levels of the house keeping protein
Pgk1 (Fig 6).
After overexpression all 6 Δelg1 mutants showed expression levels significantly higher
compared to native genomic expression, also surpassing wild type ELG1 levels by between
10%-60%.
33
Figure 6: Elg1 unstable mutations overexpression. Pgk1 was used as a loading control. The
graph represents the Western blot quantification of the relative total protein abundance of
the elg1 mutants deemed unstable, compared to the relative level of the same mutant
overexpressed from a high copy number plasmid vector pRS425. Relevant elg1 mutants
were transformed via LEU pRS425 2-micron vector into Δelg1 cells. Cells were then grown in
-LEU media prior to protein harvest. Wild type ELG1 is normalized to 100% (mean +/-
standard deviation [SD]; n= 3). *, P = 0.0427, **, P = 0.0033/0.0024, ****, P <0.0001, values
were calculated using ANOVA and Sidak’s test post-hoc analysis.
34
We then proceeded to measure chromatin bound PCNA levels in all our different mutants.
(Fig 7A). All cell cultures were grown to mid log in either YPD (or -LEU for strains carrying
high copy number plasmids), then harvested at equal titers and chromatin-bound proteins
were extracted from each culture via chromatin fractionation protocol (Materials and
Methods).
This process was repeated independently 3 times and the results were quantified after
normalizing to the protein levels of the chromatin bound protein H3 (Fig 7B). Δelg1 cells
showed the most dramatic effect, with an almost 7-fold increased amount of PCNA on the
chromatin compared to the wild type. Interface mutation TT386/7AA saw a 2-fold increase
in PCNA levels, while TT386/7DD saw a 3-fold increase. 3xSIM TT386/7AA had an increase of
almost 3-fold and 3xSIM TT386/7DD of close to 5-fold. Our novel mutation V390D increased
PCNA levels by 3.5-fold and all Walker mutations (besides KK343/4AA) showed an increase
of between 4 to 6-fold of PCNA accumulation over wild type cells.
As for our pRS425 overexpressed mutants, expression from the high copy number vectors
caused mild reductions in PCNA levels in a manner that maintained the significant change
over wild type in most of them. This confirms that the inability of these mutants to properly
unload PCNA did not stem from reduced protein levels, but from a structural difficulty of the
mutated elg1 proteins to function properly.
All chromatin-bound PCNA levels correlated with the observed phenotype trends seen in
the CRASH assay (Fig 4) as well as DNA damage sensitivity (Fig 5), further solidifying the
notion that Elg1’s PCNA unloading role is the main reason for the genomic instability effects
seen in Δelg1 cells.
35
B
****
****
****
****
****
****
****
****
****
****
**
10 *
Relative PCNA accumilation levels (%)
****
0
P
g1
pR M
D
A
34 0D
K
pe
6/ 38 D
V3 n
PR A
38 25
25
pR A
Lo pR D
D A p AA
6/ 38 25
de 25
40 , D 25
42
tio
PI
90
SI 3x TT /7A
38 TT 7D
+ DD KK /4A
7, 40 4D
K 9K
SI 3x AA /7A
D 7D
A 9A
SI
TT S 4
S4
S4
op S 4
Ty
el
4
9
40 D4 RS
40 40 RS
SI 3x
V3
le
Δ
TT M 86/
40 DD 43/
9K 40
7D 6/
9A 40
9A 40
6
3
6
ild
p
40 7,
3
40 7,
W
K
M
38 TT
0
7
K
7
TT IM
3x
D
SI
7,
7,
4
D
D
D
3/
M
D
D
D
34
3x
3x
34 KK
D
4D
3/
K
K
Figure 7: PCNA chromatin levels for the different Elg1 mutations. (A) A fractionation assay
followed by western blot to detect PCNA levels on the chromatin in the various elg1 mutant.
Histone H3 served as chromatin bound as well as loading control. (B) At least 3 experiments
were used for quantitation and the error bars represent the standard error of the mean. The
graph represents the Western blot quantification of the relative chromatin bound PCNA
abundance of the different Elg1 mutants in the cell, compared to the relative level of Wild
Type ELG1 cells that is normalized to 100% (mean +/- standard deviation [SD]; n= 3). *, P =
0.0195, **, P = 0.0032, ****, P <0.0001, values were calculated using ANOVA and Dunnett’s
test post-hoc analysis.
36
4.2 CHAPTER 2:
4.2.1 Deletion of Elg1 increases cell viability in the absence of cohesion subunit Pds5
During cell division, sister chromatids are held together until their timely separation at
anaphase. This process termed sister chromatid cohesion (SCC) is maintained by the cohesin
complex(103). One of the proteins that make up cohesin is Pds5, which is essential for
life(104), but its role is not yet fully understood. Deletion of ELG1 can partially suppress the
lethality of a temperature sensitive pds5-1 mutation but not a complete deletion of PDS5
(105). Our lab has carried out two genetic screens for high-copy number suppressors and for
spontaneously arising mutants that allowed viability for Δpds5 Δelg1 strains (106). At the
heart of the screens was a strain with genomic deletions of both PDS5 and ELG1 that had
instead a URA3 -marked covering plasmid containing another copy of PDS5. These plasmids
carried also an additional marker (either LEU2 or TRP1). Since the PDS5 gene is essential,
these strains cannot lose the URA3 containing covering plasmid and do not grow on 5-
fluoroorotic acid (5-FOA) media, which selects for Ura− cells (106)
The first screen for spontaneously arising mutants included plating the double mutant in
large numbers on 5-FOA plates and looking for colonies that can grow. After confirming
through plating on -LEU media that the covering PDS5 plasmid was truly lost, whole genome
sequencing was performed to identify the suppressor mutations in the genome (Fig 8A).
The second screen for high-copy number suppressors included the transformation of a yeast
genomic library overexpressed from a 2um plasmid marked with a LEU2 marker into our
Δpds5 Δelg1 strains bearing the PDS5 URA3 TRP1 plasmid. Yeast cells were then plated on 5-
FOA again to search for cells able to lose the URA3 plasmid. Colonies that grew were
identified to be 5-FOA resistant Leu+ Ura- and Trp- , and then the LEU2 marked library
plasmid was isolated from them and sequenced (Fig 8B)
37
A B
Figure 8: Screen for suppressors of the Δpds5 Δelg1 double mutant. (A)Illustration of the
spontaneous suppressor screen looking for mutants able to grow in the complete absence
of PDS5 and ELG1. (B) Illustration of the high-copy-number suppressor screen looking for
mutants able to grow in the complete absence of PDS5 and ELG1
The results yielded two scenarios in which Δpds5 Δelg1 strains achieved viability: the first
one was through the deletion of the G1 cyclin Cln2 (Δpds5 Δelg1 Δcln2) and the second
through over expression of the kleisin cohesin subunit Mcd1. Importantly, neither mutations
in CLN2 nor overexpression of Mdc1 were able to allow growth of a Δpds5 strain, with a
functional ELG1 gene.
Cells deleted for PDS5 are known to be defective in establishing sister chromatid cohesion,
as well as in maintaining already established cohesion (104, 107). The triple mutant strain
Δpds5 Δelg1 Δcln2 was able to lose the covering URA3 plasmid, but that not necessarily
means that the cells recovered their ability to properly conduct sister chromatid cohesion.
For this purpose, we used the two-dot assay (described in Materials and Methods).
In short, the two-dot assay works by taking advantage of the TetO system. Tet operator
sequences are placed near the centromere of a chromosome, and GFP proteins are fused
with Tet repressor sequences that target the operators. Since the two chromatids are held
together tightly, most cells show a single, bright GFP dot. Cells with two separate GFP dots
are cells with defective cohesion as the chromatids are not properly held together. Our
38
strains carried a PDS5-AID allele, which becomes degraded rapidly upon addition of Auxin
(indole acetic acid).
Although cohesin can be loaded onto chromosomes during the entirety of the cell cycle,
cells are known to increase cohesion levels only during S phase (108). So, to test for
establishment, after growing to log phase the cells were treated with Alpha factor to
synchronize and arrest the culture at the G1 phase of the cell cycle. The cells were then
sampled and released into a Nocodazole containing media with or without Auxin to allow
them to advance through the cell cycle from G1 through S and halt at G2/M (Fig 9A, 9B). For
the maintenance assay, cultures were grown to log phase, then synchronized in G2/M with
nocodazole (after SCC establishment), and only then auxin was added. (Fig 9C, 9D).
The results for both assays showed no significant increase in 2-dot percentage in the cases
of untreated samples, which sits well with the fact that there was no Pds5 degradation (Fig
9A,9C). In the cases of the treated samples tested for establishment, the PDS5-AID strain
showed about 40% two-dot percentage, signaling significant cohesion loss. Deleting Elg1 or
Cln2 reduced the number of 2-dot cells, with the combined deletion reducing it the most (P
= 0.021) hinting at a separation of function between Elg1 and Cln2 in the Pds5-absent
cohesion pathway (Fig 9B). For the Auxin treated maintenance assay samples, PDS5- AID
showed around 60% two-dot percentage, with Elg1 having a smaller rescue effect than
Cln2(Fig 9D). These results hint that Elg1 and Cln2 play different roles in sister chromatid
cohesion, with ΔElg1 having less effect after cohesion establishment.
39
Figure 9: Testing sister chromatid cohesion through the 2-dot assay. (A) Percentage of cells
showing 2 dots in a cohesion establishment assay. Cultures were grown to log phase then
added with Alpha factor [50 ng/mL] for two hours to arrest cells in G1 (first time point) then
Alpha factor was replaced with Nocodazole [15 mg/mL] for two hours to allow the cells to
advance and hold at G2/M (second time point). (B) Same, with Auxin [300 mM] added
together with the alpha factor (before S phase) (mean +/- SD; n=3 with 200 cells per strain
and experiment) *, P=0.021 was calculated using t test (C) Percentage of cells showing 2
dots in a cohesion maintenance assay. Cultures were allowed to synchronize in G2/M before
the percentage of cells with 2 dots scored. (mean +/- SD; n= 3, with 200 cells per strain and
experiment) (D) Same, with Auxin added in G2/M, after SCC was already established. *,
P=0.022 was calculated using t test.
40
4.2.2 Δpds5 Δcln2 suppression is enhanced by PCNA accumulation on chromatin in elg1 mutants
To further investigate Elg1’s role in the suppression phenotype of Δpds5, we went back to
our plasmid library containing Elg1 mutations. Given the strong correlation we witnessed
when we compared the different mutations' silencing loss phenotype to MMS sensitivity
that indicated PCNA levels affected both similarly, we tested whether a similar correlation
was seen when trying to complement Δelg1’s role on 5-FOA. A Δpds5 Δelg1 Δcln2 strain
already containing the CEN PDS5 URA3 plasmid was now transformed with our CEN ELG1
LEU2 mutant plasmids, and new cells were plated on SD-Leu-Ura, SD-Leu+5-Foa, and SD-
complete containing different MMS concentrations (Fig 10A). Alleles of ELG1 able to
complement elg1’s effect on SCC should not be viable on 5-FOA, which selects against the
covering URA3-carrying plasmid (as they will effectively be Δpds5 Δcln2) while growing well
on MMS (as they complement Δelg1's sensitivity to MMS). In contrast, mutants lacking the
ability to function as wildtype ELG1 (hence unloading PCNA) should grow on 5-FOA and
show sensitivity to MMS. Among all the mutants tested this way, the 8 that showed lack of
ability to function as wild type ELG1 grew on 5-FOA plates and showed increased MMS
sensitivity and silencing loss. The elg1 mutant KK343,344AA on the other hand, performed
as wildtype ELG1 (complementing Δelg1’s role in the triple deletion rescue phenotype) and
did not grow on 5-FOA while not showing any MMS sensitivity, matching our expectations
(Fig 10A). To further solidify our claim that it is the level of PCNA on the chromatin that
affects all phenotypes discussed, we transformed our Δpds5 Δelg1 Δcln2 strain with the
PCNA disassembly prone mutants shown before. As expected, Δpds5 Δelg1 Δcln2 cells with
self-unloading PCNA were not able to show the rescue phenotype and also lost the MMS
sensitivity typical of Δelg1 cells (Fig 10B). These results support the notion that the
phenotype of Δelg1 in relation to sister chromatid cohesion is due to accumulation of PCNA
on the DNA as well.
41
Figure 10: PCNA levels on the chromatin complement ΔPds5 suppression phenotype. (A)
Serial five-fold dilutions of yeast cultures on SD-Leu-Ura, SD+5-FOA – Leu, or SD-Complete
with methyl methane sulphonate (MMS) in the indicated concentration shows Δpds5 Δelg1
Δcln2 strain carrying CEN PDS5 URA plasmid and a CEN LEU plasmid containing different
ELG1 mutations. (B) Δpds5 Δelg1 Δcln2 strain carrying CEN PDS5 URA plasmid and genomic
disassembly prone PCNA mutations.
42
4.2.3 ∆elg1’s contribution to rescue the SCC phenotype of pds5 is a possible result of Srs2
recruitment to the chromatin by SUMOylated PCNA
After establishing that PCNA accumulation on the DNA is the cause behind the rescue effect
∆elg1 has on pds5, we wanted to know whether PCNA modifications also play a part. PCNA
is known to be able to go through different post-translational modifications on specific
residues, which affect the cellular response to DNA damage (26). If the replisome
encounters a DNA lesion PCNA can undergo mono-ubiquitination at position K164 by the
ubiquitin activating and conjugating enzymes Rad6 and Rad18. This modification leads to
the recruitment of special DNA damage tolerant polymerases that interact with PCNA
through their PIP or UB (ubiquitin binding) domains and opens a mutagenic replication
pathway called Translesion Synthesis or TLS. Alternatively, if the initial K164 conjugated
ubiquitin goes through additional ubiquitination rounds (polyubiquitination by Mms2,
Ubc13 and Rad5) different DNA polymerases will be recruited and a different non mutagenic
pathway will open, called Error-free bypass or Template switch (8). PCNA can also be added
with a SUMO group at Lysines 127 and 164 by the SUMO ligase Siz1. SUMOylation can
recruit the helicase Srs2, which has been shown to work as an anti-recombination agent by
evicting the Rad51 nucleoprotein, known to be crucial for the recombination process (109).
We replaced PCNA in our Δpds5 Δelg1 Δcln2 strain with a version that carries Arginines
instead of Lysines at positions 127 and 164 and thus cannot be modified (Fig 11A). Among
the PCNA mutants tested pol30-K164R and pol30-K127R K164R abolished Δelg1’s rescue
phenotype, hinting that modified PCNA that gets accumulated on the chromatin is
necessary to promote the suppression. Further work done to determine which of the PCNA
modifications is the relevant one done in our recently published paper (106) showed how
Δpds5 Δelg1 Δcln2 cells deleted for Rad6 or Rad18 were still able to grow on 5-FOA ,
whereas deletion of the SUMO ligase Siz1 resulted in cells no longer able to grow on 5-FOA.
This result shows that accumulation of SUMOylated PCNA is the cohesion-promoting force
at play in the Δelg1 cells. This hint led us to delete SRS2 in our Δpds5 Δelg1 Δcln2 strain, as
Srs2 is the other protein (in addition to Elg1) known to be recruited by SUMOylated PCNA.
The quadruple mutant was again unviable on 5-FOA (Fig 11B), meaning the absence of SRS2
reverses the rescue phenotype contributed by PCNA and SUMO-PCNA accumulation on the
DNA in the absence of Pds5. Furthermore, we’ve discovered that deleting the gene RAD51, a
43
substrate of Srs2 and a single strand DNA binding protein also involved in homologous
recombination, negates the effect of Δsrs2 and renders the cells viable again [as a quintuple
Δpds5 Δelg1 Δcln2 Δsrs2 Δrad51 mutant; (Fig 11B)]. In conclusion, the lack of PCNA
unloading caused by Δelg1 leads to excessive accumulation of PCNA and SUMO-PCNA on
the DNA, which in turn recruits the protein Srs2, which removes Rad51 from single stranded
DNA resulting in more exposed single strand DNA for cohesin molecules to be deposited on
in the absence of PDS5. This result implies that Pds5 plays a role in the deposition of
cohesin; in its absence, its function can be replaced by increased Srs2-driven eviction of
proteins from the ssDNA (facilitated by the increased levels of SUMOylated PCNA). In
addition, the level of Mcd1 in the cells needs to be increased, as these mutants have lost the
second function of Pds5, which is to protect Mcd1 from degradation. Deleting the CLN2
gene or directly overexpressing Mcd1 fulfil this function.
44
Figure 11: SUMO-PCNA levels and SRS2 complement ΔPds5 suppression phenotype. (A)
Serial five-fold dilutions of yeast cultures on SD-Ura and SD+5-FOA shows Δpds5 Δelg1 Δcln2
strains carrying CEN PDS5 URA plasmid as well as genomic mutations in known modification
residues of PCNA. (B) Δpds5 Δelg1 Δcln2 strain carrying CEN PDS5 URA plasmid as we as
genomic deletions of SRS2 and RAD51
45
5. Discussion
5.1 CHAPTER 1:
Elg1-mediated PCNA unloading as a driving force behind Δelg1 silencing and DNA repair phenotypes
DNA replication is a central process in the cell, and it affects other, related processes such as
DNA repair, epigenetic memory and sister chromatid cohesion. By now, the importance of
PCNA for all these processes is quite clear. PCNA acts as a coordination platform that directs
proteins to the replication fork, be they proteins directly related to replication and DNA
repair such as different DNA polymerases or proteins related to chromatin remodeling such
as protein chaperones. The combined work of loading PCNA by RFC and unloading PCNA by
the Elg1-RLC provides the cell with the ability to recycle PCNA quickly and accurately to and
from needed areas during replication. PCNA and Rfc1-5 are essential proteins, as without
them no DNA replication is possible. Elg1 on the other hand is a non-essential gene, and
cells lacking Elg1 complete DNA replication only slightly slower than wild type cells (29).
Using the CRASH assay makes possible the ability to witness even transient loss events of
epigenetic silencing at the silent mating cassettes of yeast, and it revealed how the deletion
of Elg1 leads to a high rate of transient loss of heterochromatin silencing (99).
The absence of Elg1, accompanied by both loss of silencing and increased sensitivity to DNA
Damage shown here (Figs 1,4,5) as well as other reported phenotypes of genomic stability
may be in part due to systematic inability to recycle PCNA to new needed sites after its
initial loading, causing many proteins that require PCNA’s signaling ability to localize to the
DNA, specifically at replication forks and new DNA, to perform at reduced effectivity (111,
112). One such proteins is the chromatin remodeler CAF1, which has a known interaction
site with PCNA (70, 71) , and has been shown using CRASH, to be able to compensate for the
silencing loss caused by Δelg1 when overexpressed (99). The rescue in silencing loss seen by
all disassembly prone PCNA mutants tested: D150E, E134K, S152P and V180D (Fig 1D)
further supports the notion that PCNA must maintain a certain level of dynamic localization,
provided here by steady loading by RFC and increased self-disassociation, to maintain the
ability of PCNA and PCNA-bound proteins to transition from nascent replicated DNA back to
sites of active DNA replication.
46
To further understand the connection between Elg1’s unloading activity, PCNA retention on
the chromatin, silencing control and DNA damage sensitivity we put our series of Elg1
mutants through the different assays. Elg1’s reported PIP motif as well as 3 SIM motifs had
non-significant effects on silencing control and MMS sensitivity and accordingly, also no
significant increase on PCNA retention levels (Figs 4,5,7). The Threonines at Elg1’s central
domain, predicted to possibly be located at an interaction site with PCNA (42), when
mutated to Alanines (TT386/7AA) did not have a significant effect in our CRASH and MMS
assays, and accordingly no increased PCNA retention. The combination of SIM mutations
with TT386/7 mutations did bring about an increase in all 3 phenotypes (CRASH, MMS and
PCNA retention) to an intermediate level for the Alanine case (TT386/7AA) and to a deletion
like level in the CRASH and MMS for the Aspartic acid case (TT386/7DD). This supports the
notion that the SIM motifs has importance not only for Elg1’s interaction with the SUMO
group of PCNA, but for the interaction with PCNA, which could be due to an overall change
in Elg1’s affinity to PCNA. Our novel mutation site V390 did not show an increase in silencing
loss or MMS sensitivity as well PCNA accumulation when mutated to Alanine, and a
significant increase in CRASH and MMS when mutated to Aspartic acid similar to
TT386/7DD. V390 was created based on homology with a part of the protein Yku80, that is
part of a complex crucial for telomere binding, non-homologous end joining DNA repair, as
well as telomerase activation(113). Deletion of Elg1 leads to elongated telomeres while
deletion of Yku80 to short telomers. Previous work done in our lab has shown that mutation
elg1-V390E had the ability to bring about telomere elongation, Glutamic acid is a large
amino acid of positive charge, with the potential to interrupt the connection of Elg1 with
PCNA in a way similar to Aspartic acid which is also large and positive. Importantly, V390 is
only two residues away from the threonines 386-387, and thus may also affect the interface
between Elg1 and PCNA. It is thus unlikely that the homology patch is located at a place of
protein-protein interaction between Elg1 and PCNA and that the homology with Yku80
exists due to more than chance. We also mutated the N-terminus region Δ290-319 of the
unstructured loop region containing a hydrophobic patch conserved throughout Elg1
orthologs but not in other Rfc1-like proteins. This mutation did not bring about any change
in PCNA retention levels as well as in the CRASH and MMS assays, leaving the mutants able
to function at wild type levels. The role of this conserved loop thus remains mysterious.
47
All 5 Rfc units of the PCNA loader RFC contain Walker-type ATPase motifs used for ATP
binding and hydrolysis, but both Elg1 and its mammalian ortholog ATAD5 have Walker A
motifs that deviate from consensus. The consensus motif at the Walker A domain is usually
GKT while in Elg1 it is GKKT. Thus, the role of ATP hydrolysis, assumed to be important for
clamp unloading, is unclear (114).
Walker A mutation KK343/4AA had no effect on PCNA accumulation and accordingly no

observed effect through our CRASH and MMS assays, while KK343/4DD showed significant
increased sensitivities similarly to TT386/7DD and V390D. This supports the current notion
that Elg1 has no active ATPase domain. Walker B mutations DVD407/9KVK and
DVD407/9AVA both saw significant increases in silencing loss and MMS sensitivity
correlating with PCNA levels. The anomaly of DVD407/9AVA having an effect does stand
out. Alanine is used in our mutagenesis because of its non-bulky, chemically inert, methyl
functional group that nevertheless mimics the secondary structure preferences that many of
the other amino acids possess. This overall result does not stand with current knowledge of
Elg1’s structure, but the possibility of damaging an existing hydrolysis position, hindering
PCNA recycling does exist.
Overall, the correlation between PCNA retention levels among the different Elg1 mutants
and CRASH and MMS assay results is extremely convincing. This correlation suggests that
the sensitivity to DNA damage and the increased silencing loss are the consequence of the
retention of PCNA on the chromatin, most likely due to recruitment of PCNA interacting
proteins in an unsynchronized and incorrect manner by the clamp unit, which harms the
ability of these proteins to decide between different DNA repair mechanisms and chromatin
remodeling pathways.
48
5.2 CHAPTER 2:
Deletion of Elg1 promotes SCC through SUMO-PCNA accumulation & Srs2 recruitment
The process of cell division cannot be completed without secure and accurate chromosome
segregation, which requires that the two newly synthesized DNA molecules remain
juxtaposed until Anaphase (Sister Chromatid Cohesion). Like many other cellular processes,
SCC is also connected to DNA replication and many key players in replication are known to
be critical for cohesion establishment (115, 116). Deletion of the PCNA unloader Elg1 is
known to bring about increases in chromatin-bound PCNA and SUMO-PCNA levels (50).
Results from the two-dot assay showed how ELG1 affected cohesion mostly through its
establishment process and less during maintenance (Fig 9). By using our elg1 mutants’
plasmid library, we showed how the ability of different elg1 mutations to complement the
role of Δelg1 in the Δpds5 Δcln2 rescue phenotype is opposite to their ability to grow in the
presence of the DNA damaging agent MMS (Fig 10A), reflecting their ability to unload PCNA
from the DNA. Using our spontaneously disassembling PCNA mutants (DPPs) we showed
how bypassing Elg1-dependent PCNA unloading brought about a complete reversal of the
rescue conferred by the initial deletion of ELG1 (Fig 10B). These results indicate to higher
PCNA chromatin levels in the absence of Elg1 being the cause behind the suppression of
Δpds5 Δcln2’ inviability.
The DNA helicase Srs2 is recruited to the DNA by binding SUMOylated PCNA (50), and works
to evict Rad51 filaments thus exposing more single strand DNA(110, 117). Srs2 has been
shown in the past to promote SCC through either recovery from S-phase checkpoint arrest
following DNA repair or assisting in restoring chromatin configuration after DNA repair to a
form that supports cohesion(118). Our results show how deletion of SRS2, as well as
mutations that prevent PCNA SUMOylation and thus Srs2 recruitment, reverses the rescue
phenotype of Δelg1 Δpds5 Δcln2 (Fig 11A, 11B). Moreover, we show how deletion of RAD51,
a known substrate of SRS2, again reverses the effect rendering the cells viable again (Fig
11B). All this supports the notion that a possible role for Δelg1 in the Δpds5 Δcln2 rescue is
to increase SUMO-PCNA chromatin levels that in turn leads to Srs2 recruitment that
increases Rad51 eviction from the chromatin. Why would eviction of Rad51 promote SCC? A
recent in vitro assay by the Uhlmann lab(119) showed how cohesion can be loaded onto
49
double stranded DNA, but second strand entrapment requires single strand DNA. In their
model, during DNA replication cohesin is loaded onto double stranded DNA at the leading
strand as a first step, and a second step during which cohesin entraps single stranded DNA
at the lagging strand. This sits with the increased eviction of Rad51 in Elg1’s absence, that
exposes more single stranded DNA that could increase cohesion establishment. Consistent
with this is the fact that increased levels of Smc3 acetylation, a known representative of
stable cohesion(120) , are present in Δelg1 cells (121)
50
6. References
1. C. Lundin, M. North, K. Erixon, K. Walters, D. Jenssen, A. S. H. Goldman, T. Helleday, Methyl
methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA
double-strand breaks, doi:10.1093/nar/gki681.
2. A. Moretton, J. I. Loizou, Interplay between Cellular Metabolism and the DNA Damage
Response in Cancer. Cancers (Basel). 12, 2051 (2020).
3. C. J. O’Kane, E. M. Hyland, Yeast epigenetics: the inheritance of histone modification states.

Biosci Rep. 39 (2019), doi:10.1042/BSR20182006.
4. V. Measday, P. C. Stirling, Navigating yeast genome maintenance with functional genomics.

Brief Funct Genomics. 15, 119–129 (2016).
5. C. E. Bansbach, D. Cortez, Defining genome maintenance pathways using functional genomic

approaches. Crit Rev Biochem Mol Biol. 46, 327–341 (2011).
6. V. Jackson, Deposition of newly synthesized histones: hybrid nucleosomes are not tandemly
arranged on daughter DNA strands. Biochemistry. 27, 2109–2120 (1988).
7. M. Shogren-Knaak, H. Ishii, J.-M. Sun, M. J. Pazin, J. R. Davie, C. L. Peterson, Histone H4-K16

acetylation controls chromatin structure and protein interactions. Science. 311, 844–7 (2006).
8. W. Zhang, Z. Qin, X. Zhang, W. Xiao, Roles of sequential ubiquitination of PCNA in DNA-

damage tolerance, doi:10.1016/j.febslet.2011.04.044.
9. G. Zheng, M. Kanchwala, C. Xing, H. Yu, MCM2–7-dependent cohesin loading during S phase

promotes sister-chromatid cohesion. Elife. 7 (2018), doi:10.7554/eLife.33920.
10. V. Tullio, Yeast Genomics and Its Applications in Biotechnological Processes: What Is Our
Present and Near Future? Journal of Fungi. 8, 752 (2022).
11. D. K. Breslow, D. M. Cameron, S. R. Collins, M. Schuldiner, J. Stewart-Ornstein, H. W.

Newman, S. Braun, H. D. Madhani, N. J. Krogan, J. S. Weissman, A comprehensive strategy
enabling high-resolution functional analysis of the yeast genome. Nat Methods. 5, 711–718
(2008).
12. Z. Li, F. J. Vizeacoumar, S. Bahr, J. Li, J. Warringer, F. S. Vizeacoumar, R. Min, B. VanderSluis, J.

Bellay, M. DeVit, J. A. Fleming, A. Stephens, J. Haase, Z.-Y. Lin, A. Baryshnikova, H. Lu, Z. Yan,
K. Jin, S. Barker, A. Datti, G. Giaever, C. Nislow, C. Bulawa, C. L. Myers, M. Costanzo, A.-C.
Gingras, Z. Zhang, A. Blomberg, K. Bloom, B. Andrews, C. Boone, Systematic exploration of
essential yeast gene function with temperature-sensitive mutants. Nat Biotechnol. 29, 361–
367 (2011).
13. C. López-Otín, M. A. Blasco, L. Partridge, M. Serrano, G. Kroemer, The Hallmarks of Aging.

Cell. 153, 1194–1217 (2013).
14. D. Hanahan, R. A. Weinberg, Hallmarks of Cancer: The Next Generation. Cell. 144, 646–674
(2011).
15. R. E. Johnson, R. Klassen, L. Prakash, S. Prakash, A Major Role of DNA Polymerase δ in

Replication of Both the Leading and Lagging DNA Strands. Mol Cell. 59, 163–175 (2015).
51
16. R. E. Georgescu, G. D. Schauer, N. Y. Yao, L. D. Langston, O. Yurieva, D. Zhang, J. Finkelstein,
M. E. O’donnell, Reconstitution of a eukaryotic replisome reveals suppression mechanisms
that define leading/lagging strand operation, doi:10.7554/eLife.04988.001.
17. K.-Y. Lee, S. H. Park, Eukaryotic clamp loaders and unloaders in the maintenance of genome
stability, doi:10.1038/s12276-020-00533-3.
18. A. A. Franco, W. M. Lam, P. M. Burgers, P. D. Kaufman, Histone deposition protein Asf1

maintains DNA replisome integrity and interacts with replication factor C. Genes Dev. 19,
1365–1375 (2005).
19. L. A. Anderson, N. D. Perkins, The large subunit of replication factor C interacts with the
histone deacetylase, HDAC1. Journal of Biological Chemistry. 277, 29550–29554 (2002).
20. G. L. Moldovan, B. Pfander, S. Jentsch, PCNA, the Maestro of the Replication Fork. Cell. 129
(2007), pp. 665–679.
21. C. Hoege, B. Pfander, G.-L. Moldovan, G. Pyrowolakis, S. Jentsch, “RAD6-dependent DNA

repair is linked to modification of PCNA by ubiquitin and SUMO” (2002), (available at
www.nature.com/nature).
22. D. Branzei, I. Psakhye, DNA damage tolerance. Curr Opin Cell Biol. 40 (2016), pp. 137–144.
23. M. Arbel, A. Bronstein, S. Sau, B. Liefshitz, M. Kupiec, Access to PCNA by Srs2 and Elg1
Controls the Choice between Alternative Repair Pathways in Saccharomyces cerevisiae
(2020), doi:10.1128/mBio.00705-20.
24. M. Sakato, Y. Zhou, M. M. Hingorani, ATP binding and hydrolysis-driven rate-determining

events in the RFC-catalyzed PCNA clamp loading reaction. J Mol Biol. 416, 176–191 (2012).
25. J. Majka, P. M. J. Burgers, “The PCNA-RFC Families of DNA Clamps and Clamp Loaders”
(2004).
26. M. Kupiec, Alternative clamp loaders/unloaders. FEMS Yeast Res. 16, 84 (2016).
27. C. Johnson, V. K. Gali, T. S. Takahashi, T. Kubota, PCNA Retention on DNA into G2/M Phase
Causes Genome Instability in Cells Lacking Elg1. Cell Rep. 16, 684–695 (2016).
28. S. Ben-Aroya, A. Koren, B. Liefshitz, R. Steinlauf, M. Kupiec, “ELG1, a yeast gene required for
genome stability, forms a complex related to replication factor C,” (available at
www.pnas.orgcgidoi10.1073pnas.1633757100).
29. T. Kubota, K. Nishimura, M. T. Kanemaki, A. D. Donaldson, The Elg1 Replication Factor C-like
Complex Functions in PCNA Unloading during DNA Replication. Mol Cell. 50, 273–280 (2013).
30. S. Ben-Aroya, A. Koren, B. Liefshitz, R. Steinlauf, M. Kupiec, “ELG1, a yeast gene required for
genome stability, forms a complex related to replication factor C,” (available at
31. S. Smith, J.-Y. Hwang, S. Banerjee, A. Majeed, A. Gupta, K. Myung, “Mutator genes for
suppression of gross chromosomal rearrangements identified by a genome-wide screening in
Saccharomyces cerevisiae,” (available at www.pnas.orgcgidoi10.1073pnas.0403093101).
52
32. D. T. Scholes, M. Banerjee, B. Bowen, M. J. Curcio, “Multiple Regulators of Ty1 Transposition
in Saccharomyces cerevisiae Have Conserved Roles in Genome Maintenance,” (available at
https://academic.oup.com/genetics/article/159/4/1449/6049793).
33. H. Ogiwara, A. Ui, T. Enomoto, M. Seki, Role of Elg1 protein in double strand break repair,
doi:10.1093/nar/gkl1027.
34. S. H. Askree, T. Yehuda, S. Smolikov, R. Gurevich, J. Hawk, C. Coker, A. Krauskopf, M. Kupiec,

M. J. Mceachern, “A genome-wide screen for Saccharomyces cerevisiae deletion mutants
that affect telomere length” (2004), (available at
35. S. Smolikov, Y. Mazor, A. Krauskopf, “ELG1, a regulator of genome stability, has a role in
telomere length regulation and in silencing” (2004), (available at
36. M. B. Davidson, G. W. Brown, The N- and C-termini of Elg1 contribute to the maintenance of
genome stability. DNA Repair (Amst). 7, 1221–1232 (2008).
37. D. Alvaro, M. Lisby, R. Rothstein, Genome-Wide Analysis of Rad52 Foci Reveals Diverse
Mechanisms Impacting Recombination, doi:10.1371/journal.pgen.0030228.eor.
38. I. Gazy, B. Liefshitz, O. Parnas, M. Kupiec, Elg1, a central player in genome stability. Mutation
Research/Reviews in Mutation Research. 763, 267–279 (2015).
39. I. Gazy, B. Liefshitz, A. Bronstein, O. Parnas, N. Atias, R. Sharan, M. Kupiec, A Genetic Screen
for High Copy Number Suppressors of the Synthetic Lethality Between elg1D and srs2D in
Yeast (2013), doi:10.1534/g3.113.005561.
40. O. Parnas, A. Zipin-Roitman, Y. Mazor, B. Liefshitz, S. Ben-Aroya, The Elg1 Clamp Loader Plays
a Role in Sister Chromatid Cohesion. PLoS One. 4, 5497 (2009).
41. B. Ho, A. Baryshnikova, G. W. Brown, Unification of Protein Abundance Datasets Yields a

Quantitative Saccharomyces cerevisiae Proteome. Cell Syst. 6, 192-205.e3 (2018).
42. K. Shemesh, M. Sebesta, M. Pacesa, S. Sau, A. Bronstein, O. Parnas, B. Liefshitz, ˇ Ceslovas

Venclovas, L. Krejci, M. Kupiec, A structure-function analysis of the yeast Elg1 protein reveals
the importance of PCNA unloading in genome stability maintenance. Nucleic Acids Res. 45,
3189–3203 (2017).
43. A. Chiraniya, J. Finkelstein, M. Odonnell, L. B. Bloom, A Novel Function for the Conserved
Glutamate Residue in the Walker B Motif of Replication Factor C. Genes (Basel). 4, 134–151
(2013).
44. K. E. Duderstadt, J. M. Berger, AAA+ ATPases in the Initiation of DNA Replication. Crit Rev
Biochem Mol Biol. 43, 163–187 (2008).
45. T. Kubota, M. Kyungjae, A. D. Donaldson, Cell Cycle Is PCNA unloading the central function of
the Elg1/ ATAD5 replication factor C-like complex? (2013), doi:10.4161/cc.25626.
46. O. Kerscher, SUMO junction - What’s your function? New insights through SUMO-interacting
motifs. EMBO Rep. 8 (2007), pp. 550–555.
53
47. A. Minty, X. Dumont, M. Kaghad, D. Caput, Covalent modification of p73α by SUMO-1: Two-
hybrid screening with p73 identifies novel SUMO-1-interacting proteins and a SUMO-1
interaction motif. Journal of Biological Chemistry. 275, 36316–36323 (2000).
48. J. Song, L. K. Durrin, T. A. Wilkinson, T. G. Krontiris, Y. Chen, “Identification of a SUMO-binding

motif that recognizes SUMO-modified proteins” (2004), (available at
49. E. Warbrick, PCNA binding through a conserved motif. BioEssays. 20, 195–199 (1998).
50. O. Parnas, A. Zipin-Roitman, B. Pfander, B. Liefshitz, Y. Mazor, S. Ben-Aroya, S. Jentsch, M.

Kupiec, Elg1, an alternative subunit of the RFC clamp loader, preferentially interacts with
SUMOylated PCNA. EMBO Journal. 29, 2611–2622 (2010).
51. T. Kubota, Y. Katou, R. Nakato, K. Shirahige, A. D. Donaldson, Replication-Coupled PCNA

Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment
Ligation. Cell Rep. 12, 774–787 (2015).
52. C. Yu, H. Gan, J. Han, Z. X. Zhou, S. Jia, A. Chabes, G. Farrugia, T. Ordog, Z. Zhang, Strand-
Specific Analysis Shows Protein Binding at Replication Forks and PCNA Unloading from
Lagging Strands when Forks Stall. Mol Cell. 56, 551–563 (2014).
53. G. Schäfer, E. M. Smith, H. G. Patterton, The Saccharomyces cerevisiae linker histone Hho1p,
with two globular domains, can simultaneously bind to two four-way junction DNA
molecules. Biochemistry. 44, 16766–16775 (2005).
54. I. Lacal, R. Ventura, T. J. Stevenson, A. J. Hannan, Epigenetic Inheritance: Concepts,

Mechanisms and Perspectives (2018), doi:10.3389/fnmol.2018.00292.
55. P. S. Kayne, U. J. Kim, M. Han, J. R. Mullen, F. Yoshizaki, M. Grunstein, Extremely conserved

histone H4 N terminus is dispensable for growth but essential for repressing the silent mating
loci in yeast. Cell. 55, 27–39 (1988).
56. P. Laurenson, J. Rine, “Silencers, Silencing, and Heritable Transcriptional States” (1992),
(available at https://journals.asm.org/journal/mr).
57. J. E. Brownell, J. Zhou, T. Ranalli, “Tetrahymena Histone Acetyltransferase A: A Homolog to

Yeast Gcn5p Linking Histone Acetylation to Gene Activation” (1996).
58. F. W. Symington, J. Sprent, ; M Sarmiento, A. L. Glasebrook, F. W. Fitch, “RL72 (IgM antibodies

to mouse CD4), 31 M (IgM anti-bodies to mouse CD8), and BP-1 07 (antibodies to mouse
MHC class 11 Ab) A Mammalian Histone Deacetylase Related to the Yeast Transcriptional
Regulator Rpd3p” (1985), (available at https://www.science.org).
59. G. J. Hoppe, J. C. Tanny, A. D. Rudner, S. A. Gerber, S. Danaie, S. P. Gygi, D. Moazed, Steps in

Assembly of Silent Chromatin in Yeast: Sir3-Independent Binding of a Sir2/Sir4 Complex to
Silencers and Role for Sir2-Dependent Deacetylation. Mol Cell Biol. 22, 4167–4180 (2002).
60. Z. Zhang, M. K. Hayashi, O. Merkel, B. Stillman, R. M. Xu, Structure and function of the BAH-
containing domain of Orc1p in epigenetic silencing. EMBO Journal. 21, 4600–4611 (2002).
61. L. N. Rusché, A. L. Kirchmaier, J. Rine, Ordered Nucleation and Spreading of Silenced

Chromatin in Saccharomyces cerevisiae. Mol Biol Cell. 13, 2207–2222 (2002).
54
62. R. H. Morse, RAP, RAP, open up! New wrinkles for RAP1 in yeast. Trends in Genetics. 16, 51–
53 (2000).
63. S. Imai, C. M. Armstrong, M. Kaeberlein, L. Guarente, Transcriptional silencing and longevity

protein Sir2 is an NAD-dependent histone deacetylase. Nature. 403, 795–800 (2000).
64. L. Pillus, J. Rine, Epigenetic inheritance of transcriptional states in S. cerevisiae. Cell. 59, 637–
647 (1989).
65. L. Gershon, M. Kupiec, The Amazing Acrobat: Yeast’s Histone H3K56 Juggles Several
Important Roles While Maintaining Perfect Balance (2021), doi:10.3390/genes12030342.
66. A. A. Franco, W. M. Lam, P. M. Burgers, P. D. Kaufman, Histone deposition protein Asf1

maintains DNA replisome integrity and interacts with replication factor C (2005),
doi:10.1101/gad.1305005.
67. V. K. Gali, D. Dickerson, Y. Katou, K. Fujiki, K. Shirahige, T. Owen-Hughes, T. Kubotaid, A. D.

Donaldsonid, J. E. Sale, Identification of Elg1 interaction partners and effects on post-
replication chromatin re-formation (2018), doi:10.1371/journal.pgen.1007783.
68. R. Driscoll, A. Hudson, S. P. Jackson, Yeast Rtt109 Promotes Genome Stability by Acetylating
Histone H3 on Lysine 56. Science (1979). 315, 649–652 (2007).
69. D. Su, Q. Hu, Q. Li, J. R. Thompson, G. Cui, A. Fazly, B. A. Davies, M. V. Botuyan, Z. Zhang, G.
Mer, Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone
Rtt106. Nature. 482 (2012), doi:10.1038/nature10861.
70. C. M. Kondratick, J. M. Litman, K. v Shaffer, M. Todd Washington, L. M. Dieckman, Crystal

structures of PCNA mutant proteins defective in gene silencing suggest a novel interaction
site on the front face of the PCNA ring (2018), doi:10.1371/journal.pone.0193333.
71. T. R. Ben-Shahar, A. G. Castillo, M. J. Osborne, K. L. B. Borden, J. Kornblatt, A. Verreault, Two

Fundamentally Distinct PCNA Interaction Peptides Contribute to Chromatin Assembly Factor
1 Function. Mol Cell Biol. 29, 6353–6365 (2009).
72. J.-M. Peters, A. Tedeschi, J. Schmitz, The cohesin complex and its roles in chromosome
biology. Genes Dev. 22, 3089–3114 (2008).
73. J.-M. Peters, T. Nishiyama, Sister Chromatid Cohesion. Cold Spring Harb Perspect Biol. 4,
a011130–a011130 (2012).
74. A. P. Joglekar, A. J. Hunt, A Simple, Mechanistic Model for Directional Instability during
Mitotic Chromosome Movements. Biophys J. 83, 42–58 (2002).
75. K. Nasmyth, Disseminating the Genome: Joining, Resolving, and Separating Sister Chromatids
During Mitosis and Meiosis. Annu Rev Genet. 35, 673–745 (2001).
76. L. Ström, C. Sjögren, DNA Damage-Induced Cohesion. Cell Cycle. 4, 536–539 (2005).
77. C. H. Haering, J. Löwe, A. Hochwagen, K. Nasmyth, Molecular Architecture of SMC Proteins

and the Yeast Cohesin Complex. Mol Cell. 9, 773–788 (2002).
78. S. Gruber, C. H. Haering, K. Nasmyth, Chromosomal Cohesin Forms a Ring. Cell. 112, 765–777
(2003).
55
79. C. Michaelis, R. Ciosk, K. Nasmyth, Cohesins: Chromosomal Proteins that Prevent Premature
Separation of Sister Chromatids. Cell. 91, 35–45 (1997).
80. V. Guacci, D. Koshland, A. Strunnikov, A Direct Link between Sister Chromatid Cohesion and
Chromosome Condensation Revealed through the Analysis of MCD1 in S. cerevisiae. Cell. 91,
47–57 (1997).
81. P. Arumugam, S. Gruber, K. Tanaka, C. H. Haering, K. Mechtler, K. Nasmyth, ATP Hydrolysis Is

Required for Cohesin’s Association with Chromosomes. Current Biology. 13, 1941–1953
(2003).
82. C. H. Haering, D. Schoffnegger, T. Nishino, W. Helmhart, K. Nasmyth, J. Löwe, Structure and

Stability of Cohesin’s Smc1-Kleisin Interaction. Mol Cell. 15, 951–964 (2004).
83. R. Ciosk, M. Shirayama, A. Shevchenko, T. Tanaka, A. Toth, A. Shevchenko, K. Nasmyth,

Cohesin’s Binding to Chromosomes Depends on a Separate Complex Consisting of Scc2 and
Scc4 Proteins. Mol Cell. 5, 243–254 (2000).
84. A. Lengronne, J. McIntyre, Y. Katou, Y. Kanoh, K.-P. Hopfner, K. Shirahige, F. Uhlmann,

Establishment of Sister Chromatid Cohesion at the S. cerevisiae Replication Fork. Mol Cell. 23,
787–799 (2006).
85. F. Hou, H. Zou, Two Human Orthologues of Eco1/Ctf7 Acetyltransferases Are Both Required
for Proper Sister-Chromatid Cohesion. Mol Biol Cell. 16, 3908–3918 (2005).
86. D. Ivanov, A. Schleiffer, F. Eisenhaber, K. Mechtler, C. H. Haering, K. Nasmyth, Eco1 Is a Novel

Acetyltransferase that Can Acetylate Proteins Involved in Cohesion. Current Biology. 12, 323–
328 (2002).
87. J. Zhang, X. Shi, Y. Li, B.-J. Kim, J. Jia, Z. Huang, T. Yang, X. Fu, S. Y. Jung, Y. Wang, P. Zhang, S.-
T. Kim, X. Pan, J. Qin, Acetylation of Smc3 by Eco1 Is Required for S Phase Sister Chromatid
Cohesion in Both Human and Yeast. Mol Cell. 31, 143–151 (2008).
88. G.-L. Moldovan, B. Pfander, S. Jentsch, PCNA Controls Establishment of Sister Chromatid
Cohesion during S Phase. Mol Cell. 23, 723–732 (2006).
89. E. Ünal, J. M. Heidinger-Pauli, W. Kim, V. Guacci, I. Onn, S. P. Gygi, D. E. Koshland, A Molecular

Determinant for the Establishment of Sister Chromatid Cohesion. Science (1979). 321, 566–
569 (2008).
90. K.-L. Chan, T. Gligoris, W. Upcher, Y. Kato, K. Shirahige, K. Nasmyth, F. Beckouët, Pds5
promotes and protects cohesin acetylation. Proceedings of the National Academy of Sciences.
110, 13020–13025 (2013).
91. T. Hartman, K. Stead, D. Koshland, V. Guacci, Pds5p Is an Essential Chromosomal Protein

Required for Both Sister Chromatid Cohesion and Condensation in Saccharomyces cerevisiae.
Journal of Cell Biology. 151, 613–626 (2000).
92. T. Sutani, T. Kawaguchi, R. Kanno, T. Itoh, K. Shirahige, Budding Yeast Wpl1(Rad61)-Pds5

Complex Counteracts Sister Chromatid Cohesion-Establishing Reaction. Current Biology. 19,
492–497 (2009).
93. F. Uhlmann, F. Lottspeich, K. Nasmyth, “Sister-chromatid separation at anaphase onset is

promoted by cleavage of the cohesin subunit Scc1” (1999), (available at www.nature.com).
56
94. Z. Yuan, R. Georgescu, R. de L. A. Santos, D. Zhang, L. Bai, N. Y. Yao, G. Zhao, M. E. O’Donnell,
H. Li, Ctf4 organizes sister replisomes and Pol α into a replication factory. Elife. 8 (2019),
doi:10.7554/eLife.47405.
95. C. P. Samora, J. Saksouk, P. Goswami, B. O. Wade, M. R. Singleton, P. A. Bates, A. Lengronne,

A. Costa, F. Uhlmann, Ctf4 Links DNA Replication with Sister Chromatid Cohesion
Establishment by Recruiting the Chl1 Helicase to the Replisome. Mol Cell. 63, 371–384 (2016).
96. K. Tong, R. v. Skibbens, Pds5 regulators segregate cohesion and condensation pathways in
Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences. 112, 7021–7026
(2015).
97. M. E. Maradeo, R. v. Skibbens, The Elg1-RFC Clamp-Loading Complex Performs a Role in Sister
Chromatid Cohesion. PLoS One. 4, e4707 (2009).
98. G. Schlissel, M. K. Krzyzanowski, F. Caudron, Y. Barral, J. Rine, Aggregation of the Whi3

protein, not loss of heterochromatin, causes sterility in old yeast cells. Science (1979). 355,
1184–1187 (2017).
99. R. Janke, G. A. King, M. Kupiec, J. Rine, Pivotal roles of PCNA loading and unloading in
heterochromatin function. Proc Natl Acad Sci U S A. 115, E2030–E2039 (2018).
100. C. Johnson, V. K. Gali, T. S. Takahashi, T. Kubota, PCNA Retention on DNA into G2/M Phase
Causes Genome Instability in Cells Lacking Elg1. Cell Rep. 16, 684–695 (2016).
101. C. ˇ E. Venclovas, M. E. Colvin, M. P. Thelen, Molecular modeling-based analysis of

interactions in the RFC-dependent clamp-loading process (2002), doi:10.1110/ps.0214302.
102. M.-S. Kang, E. Ryu, S.-W. Lee, J. Park, N. Y. Ha, J. S. Ra, Y. J. Kim, J. Kim, M. Abdel-Rahman, S.
H. Park, K.-Y. Lee, H. Kim, S. Kang, K. Myung, Regulation of PCNA cycling on replicating DNA by
RFC and RFC-like complexes, doi:10.1038/s41467-019-10376-w.
H. Park, K.-Y. Lee, H. Kim, S. Kang, K. Myung, Regulation of PCNA cycling on replicating DNA by
RFC and RFC-like complexes, doi:10.1038/s41467-019-10376-w.
104. J. M. Peters, T. Nishiyama, Sister chromatid cohesion. Cold Spring Harb Perspect Biol. 4
(2012), doi:10.1101/cshperspect.a011130.
105. N. Zhang, L. E. Coutinho, D. Pati, Molecular Sciences PDS5A and PDS5B in Cohesin Function
and Human Disease (2021), doi:10.3390/ijms22115868.
106. M. E. Maradeo, R. v Skibbens, Replication Factor C Complexes Play Unique Pro-and Anti-
Establishment Roles in Sister Chromatid Cohesion. PLoS One. 5, 15381 (2010).
107. K. Choudhary, Z. Itzkovich, E. Alonso-Perez, H. Bishara, B. Dunn, G. Sherlock, M. Kupiec, S.

cerevisiae Cells Can Grow without the Pds5 Cohesin Subunit. mBio. 13 (2022),
doi:10.1128/mbio.01420-22.
108. S. Vaur, A. Feytout, S. Vazquez, J. P. Javerzat, Pds5 promotes cohesin acetylation and stable
cohesin-chromosome interaction. EMBO Rep. 13, 645–652 (2012).
57
109. M. Srinivasan, M. Fumasoni, N. J. Petela, A. Murray, K. A. Nasmyth, Cohesion is established
during DNA replication utilising chromosome associated cohesin rings as well as those loaded
de novo onto nascent DNAs. Elife. 9 (2020), doi:10.7554/eLife.56611.
110. B. Pfander, G.-L. Moldovan, M. Sacher, C. Hoege, S. Jentsch, SUMO-modified PCNA recruits
Srs2 to prevent recombination during S phase (2005), doi:10.1038/nature03665.
111. R. Georgescu, L. Langston, M. O’Donnell, A proposal: Evolution of PCNA’s role as a marker of

newly replicated DNA. DNA Repair (Amst). 29, 4–15 (2015).
112. K. Shibahara, B. Stillman, Replication-Dependent Marking of DNA by PCNA Facilitates CAF-1-

Coupled Inheritance of Chromatin. Cell. 96, 575–585 (1999).
113. S. J. Boulton, Components of the Ku-dependent non-homologous end-joining pathway are

involved in telomeric length maintenance and telomeric silencing. EMBO J. 17, 1819–1828
(1998).
H. Park, K. Lee, H. Kim, S. Kang, K. Myung, Regulation of PCNA cycling on replicating DNA by
RFC and RFC-like complexes. Nat Commun. 10, 2420 (2019).
115. M. Arbel, K. Choudhary, O. Tfilin, M. Kupiec, PCNA Loaders and Unloaders-One Ring That
Rules Them All (2021), doi:10.3390/genes12111812.
116. M. L. Mayer, I. Pot, M. Chang, H. Xu, V. Aneliunas, T. Kwok, R. Newitt, R. Aebersold, C. Boone,
G. W. Brown, P. Hieter, Identification of Protein Complexes Required for Efficient Sister
Chromatid Cohesion. Mol Biol Cell. 15, 1736–1745 (2004).
117. V. Marini, L. Krejci, Srs2: The “Odd-Job Man” in DNA repair. DNA Repair (Amst). 9, 268–275
(2010).
118. C. D. Warren, D. M. Eckley, M. S. Lee, J. S. Hanna, A. Hughes, B. Peyser, C. Jie, R. Irizarry, F. A.

Spencer, S-Phase Checkpoint Genes Safeguard High-Fidelity Sister Chromatid Cohesion. Mol
Biol Cell. 15, 1724–1735 (2004).
119. Y. Murayama, C. P. Samora, Y. Kurokawa, H. Iwasaki, F. Uhlmann, Establishment of DNA-DNA

Interactions by the Cohesin Ring. Cell. 172, 465-477.e15 (2018).
120. T. R. Ben-Shahar, S. Heeger, C. Lehane, P. East, H. Flynn, M. Skehel, F. Uhlmann, Eco1-

Dependent Cohesin Acetylation During Establishment of Sister Chromatid Cohesion. Science
(1979). 321, 563–566 (2008).
121. H. W. Liu, C. Bouchoux, M. Panarotto, Y. Kakui, H. Patel, F. Uhlmann, Division of Labor

between PCNA Loaders in DNA Replication and Sister Chromatid Cohesion Establishment.
Mol Cell. 78, 725-738.e4 (2020).
58
‫‪ .7‬תקציר‬
‫פרק ‪1‬‬
‫בשמר האפייה ‪ ,Saccharomyces cerevisiae‬טבעת הידוק הדנ”א הזזה ‪ ,PCNA‬משחקת תפקיד‬

‫מרכזי בהעתקה ותיקון של דנ"א‪ ,PCNA .‬הנוכח על הגדיל המוביל‪ ,‬ועל כל אחד מגדילי אוקזקי‬
‫המשתרכים‪ ,‬פועל כפלטפורמת נחיתה על מנת להגביר את יכולת העבודה של פולימראזות דנ"א‬
‫שונות‪ ,‬וגם כמרכז שילוח אותות לחלבונים אחרים‪ .‬הרכבתו של ‪ PCNA‬על גבי הדנ"א מתבצעת על ידי‬
‫קומפלקס ה ‪ RFC‬ופירוקו מהדנ"א מתבצע על ידי קומפלקס ‪ RFC - Like‬המכיל את תת היחידה ‪.Elg1‬‬
‫איזורים שונים בגנום יכולים להיות במצב פתוח (אאוכרומטין) או במצב סגור המגביל ביטוי מהם‬
‫(הטרוכרומטין)‪ .‬מודיפיקציות שלאחר‪-‬תרגום על היסטונים הן שמגדירות ומבדילות בין המצבים האלו‪.‬‬
‫במשך הכפלת דנ"א‪ ,‬נוקלאוזומים מפונים מלפני מזלג ההכפלה‪ .‬התורשה של תכונות‬
‫אפיגנטיגות(זיכרון אפיגנטי) דורשת שההיסטונים בעלי המודיפיקציות הללו יעברו ארגון מחדש בקרב‬
‫שתי מולקולות הדנ"א החדשות שנוצרו לאחר מעבר מזלג ההכפלה‪ .‬למרות הארגון מחדש המאסיבי‬
‫הזה של מבנה הכרומטין‪ ,‬השתקת ביטוי גנים נשמרת לאורך שלב ‪ S‬של מחזור התא‪.‬‬
‫מוטציות בפורק ה ‪ PCNA‬הלא הוא ‪ ,Elg1‬מובילות לפנוטיפים מגוונים‪ ,‬הכוללים רגישות מוגברת‬
‫לחומרים מזיקי דנ"א‪ ,‬פגמים בלכידות הכרומטידות האחיות (‪ )SCC‬ורקומבינציה מוגברת‪ .‬הן גם‬
‫גורמות לאובדן השתקה משמעותי בלוקוסים השקטים בדר"כ ‪ HML‬ו ‪ .HMR‬המטרה שלנו הייתה‬
‫לקבוע את המנגנונים דרכם ‪ Elg1‬משפיע על זיכרון אפיגנטי‪ .‬לצורך כך בחנו סדרה של אללים שונים‬
‫של ‪ Elg1‬המכילים מוטציות ספציפיות המשפיעות על יכולת פריקת ה ‪ PCNA‬שלו‪ ,‬והשוונו את היכולת‬
‫שלהם לשמר את ההשתקה בלוקוס ה ‪ HMR‬השמרי‪ .‬מצאנו שגובה ההשפעה הנראה בשיטה הזאת‪,‬‬
‫דומה לגובה הרגישות של האללים לנזקי דנ"א‪ ,‬ושניהם נמצאו גם בקורלציה עם כמות ה ‪PCNA‬‬
‫הנותרת על הכרומטין בקרב המוטנטים השונים‪.‬‬
‫התוצאות הללו מדגימות את החפיפה והתיאום בין העתקת ותיקון דנ"א לבין עיצוב מחדש של‬
‫כרומטין‪ ,‬כמו גם את התפקידים הרב‪-‬מערכתיים של טבעת ה ‪ ,PCNA‬ואת תחומי האחריות של‬
‫הרגולטור שלו ‪.Elg1‬‬
‫‪59‬‬
‫פרק ‪2‬‬
‫במהלך הכפלת דנ"א ‪ ,‬הכרומטידות האחיות מוחזקות יחדיו על שלב האנאפאזה בו הן יופרדו‬
‫לצדדים מנוגדים בתא המתחלק‪ .‬ביצוע נכון של התהליך המכונה "לכידות כרומטידות אחיות" (‪)SCC‬‬
‫הוא חיוני להפרדה נכונה של כרומטידות אחיות בתא‪ ,‬קיפול כרומוזומים ללולאות‪ ,‬וביטוי גנים‪.‬‬
‫החלבון העיקרי מאחורי תהליך ה ‪ SCC‬הוא קומפלקס הקוהזין‪ ,‬המחזיק את הכרומטידות האחיות‬
‫יחדיו משלב ‪.S‬‬
‫קוהזין הוא קומפלקס ענק‪ ,‬המורכב משני חלבונים מוארכים הנקראים ‪ Smc1‬ו ‪ ,Smc3‬המגושרים‬
‫ביניהם על ידי תחת היחידה ‪ Mcd1/Scc2‬מסוג ‪ .kleisin‬שכבה שלישית של תתי יחידות של קוהזין‬
‫כוללת את ‪ ,Wpl/Rad61 ,Scc3/Irr1‬ו ‪ ,Pds5‬אשר באות במגע עם קוהזין דרך תת היחידה ‪kleisin‬‬
‫שלו‪ .‬תת היחידה מסוג ‪ ,Pds5‬היא חיונית לתהליך ה ‪ ,SCC‬אבל התפקיד שלה עדיין לא מובן לחלוטין‪.‬‬
‫מחיקה של הגן ‪ Elg1‬יכולה לדכא את הקטלניות של אלל רגיש טמפרטורה של ‪ Pds5‬ולהציל תאים‬
‫המכילים אותו ממוות‪ ,‬אבל היא לא יכולה להציל תאים בהם הגן ‪ Pds5‬נעדר בשלמותו‪.‬‬
‫המעבדה שלנו ביצעה שתי סריקות נפרדות מבוססות החומר ‪ ,5-FOA‬במטרה למצוא מוטציות‬
‫ספונטניות היכולות להציל תאים בעלי הגנוטיפ הקטלני ‪ .Δelg1Δpds5‬התוצאות שלנו מראות שתאים‬
‫יכולים להישאר בחיים בהיעדר ‪ Pds5‬בהינתן שני תנאים‪ :‬הראשון הוא העלאה ברמת הביטוי של הגן‬
‫‪( Mcd1‬שיכולה להתרחש דרך מוטציה בגן ‪ Cln2‬הפועל כציקלין ‪ )G1‬והשני הוא העלאה של רמות‬
‫‪ PCNA‬בעל מודיפיקצית ‪( SUMO‬נגרמת בעקבות חוסר פריקה ע"י ‪ .(Elg1‬רמת ה ‪SUMO-PCNA‬‬
‫הגבוהה מעלה בהתאמה את רמתו של ההליקאז ‪( Srs2‬דרך הצימוד שלו ל ‪ ,(SUMO-PCNA‬והוא‬
‫פועל לפנות מולקולות ‪ Rad51‬ממזלג ההכפלה שנמצא בתנועה‪ .‬הפעולה הזאת בתורה‪ ,‬משחררת‬
‫כמויות גדולות יותר של דנ"א חד גדילי שמשמש להגברת טעינת טבעות קוהזין על גבי הדנ"א‪,‬‬
‫ושיפור תהליך ה ‪.SCC‬‬
‫התוצאות האלה מדגימות את התפקיד המייצב של ‪ Pds5‬בקומפלקס הקוהזין לטובת תהליך ה ‪,SCC‬‬
‫והן גם חושפות מסלול חלופי בו החוסר בפעילות ‪ Elg1‬עוזרת לתא להתגבר על היעדר ‪.Pds5‬‬
‫‪60‬‬
‫אוניברסיטת תל אביב‬
‫הפקולטה למדעי החיים ע"ש ג'ורג' ס' וייז‬
‫בית הספר למחקר ביו‪-‬רפואי ולחקר הסרטן ע"ש שמוניס‬
‫תפקידים רגולטורים של ‪ Elg1‬פורק טבעת ה‪ ,PCNA-‬בתהליכי השתקת גנים‬

‫ובתהליך "לכידות כרומטיות אחיות" (‪ (sister chromatid cohesion‬ב‬
‫‪Saccharomyces cerevisiae‬‬
‫חיבור זה הוגש כעבודת גמר לקראת התואר "מוסמך" אוניברסיטה‬

‫במסלול גנטיקה באוניברסיטת תל‪-‬אביב‬
‫מאת‬
‫זיו איצקוביץ'‬
‫‪200834505‬‬
‫בהנחיית‬
‫פרופ' מרטין קופייק‬
‫פברואר ‪2022‬‬
‫‪61‬‬

Ziv Itzkovich MSC Thesis

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Ziv Itzkovich MSC Thesis

Uploaded by

Copyright:

Available Formats

TEL-AVIV UNIVERSITY

GEORGE S. WISE FACULTY OF LIFE SCIENCES GRADUATE SCHOOL

Regulatory roles of the Elg1 PCNA-unloader in gene silencing and

Thesis submitted for the M.Sc. degree in Genetics at Tel-Aviv University

Under the supervision of

Prof. Martin Kupiec

Different regions of the genome can be in an open, accessible state (euchromatin) or in an

Mutations in the PCNA-unloader Elg1 confer various phenotypes, including sensitivity to

2.3. The PCNA unloader Elg1

Table 1: Yeast strains – CRASH strains, PCNA disassembly prone mutants

Table 2: Yeast strains – CRASH strains, Elg1 mutants

3.2. List of plasmids

Table 1: Elg1 mutants

Number Genotype Source

pRS 415 elg1 I 28,93,121,122 AKAA TT386/7DD-13MYC ::KanMX

pRS415 elg1 KK343/4DD-13MYC::KanMX Centromeric expression

pRS415 elg1 ∆aa 282-319 replaced with linker of 5aa::GCACG -

pRS415 elg1 TT386/7AA-13myc::KanMX Centromeric expression

pRS415 elg1 I 28,93,121,122 AKAA-13 myc::KanMX Centromeric

4334 pRS415 elg1 V390A-13myc::KanMX Centromeric expression vector Kupiec lab

pRS415 elg1 KK343/4AA-9myc::KanMX Centromeric expression

pRS415 elg1 TT386/7DD-13myc::KanMX Centromeric expression

pRS415 elg1D407A,D409A -13myc::KANMX Centromeric

E. coli Plasmid extraction

Yeast chemical transformation

Chromatin fractionation assay

Cells from 50 ml cultures (OD600<1.0) were collected by centrifugation, successively

Protein Western Blot

Yeast genomic DNA extraction

Quantification of Silencing Loss by Flow Cytometry

3.4. Yeast genetics

4.1.1 The role of ELG1-mediated PCNA unloading in heterochromatin formation

To further investigate the relationship between Elg1–RLC-mediated PCNA unloading and

Walker A mutation KK343/4AA had no effect on PCNA accumulation and accordingly no

3. C. J. O’Kane, E. M. Hyland, Yeast epigenetics: the inheritance of histone modification states.

4. V. Measday, P. C. Stirling, Navigating yeast genome maintenance with functional genomics.

5. C. E. Bansbach, D. Cortez, Defining genome maintenance pathways using functional genomic

7. M. Shogren-Knaak, H. Ishii, J.-M. Sun, M. J. Pazin, J. R. Davie, C. L. Peterson, Histone H4-K16

8. W. Zhang, Z. Qin, X. Zhang, W. Xiao, Roles of sequential ubiquitination of PCNA in DNA-

9. G. Zheng, M. Kanchwala, C. Xing, H. Yu, MCM2–7-dependent cohesin loading during S phase

11. D. K. Breslow, D. M. Cameron, S. R. Collins, M. Schuldiner, J. Stewart-Ornstein, H. W.

12. Z. Li, F. J. Vizeacoumar, S. Bahr, J. Li, J. Warringer, F. S. Vizeacoumar, R. Min, B. VanderSluis, J.

13. C. López-Otín, M. A. Blasco, L. Partridge, M. Serrano, G. Kroemer, The Hallmarks of Aging.

15. R. E. Johnson, R. Klassen, L. Prakash, S. Prakash, A Major Role of DNA Polymerase δ in

18. A. A. Franco, W. M. Lam, P. M. Burgers, P. D. Kaufman, Histone deposition protein Asf1

21. C. Hoege, B. Pfander, G.-L. Moldovan, G. Pyrowolakis, S. Jentsch, “RAD6-dependent DNA

24. M. Sakato, Y. Zhou, M. M. Hingorani, ATP binding and hydrolysis-driven rate-determining

34. S. H. Askree, T. Yehuda, S. Smolikov, R. Gurevich, J. Hawk, C. Coker, A. Krauskopf, M. Kupiec,

41. B. Ho, A. Baryshnikova, G. W. Brown, Unification of Protein Abundance Datasets Yields a

42. K. Shemesh, M. Sebesta, M. Pacesa, S. Sau, A. Bronstein, O. Parnas, B. Liefshitz, ˇ Ceslovas

48. J. Song, L. K. Durrin, T. A. Wilkinson, T. G. Krontiris, Y. Chen, “Identification of a SUMO-binding

50. O. Parnas, A. Zipin-Roitman, B. Pfander, B. Liefshitz, Y. Mazor, S. Ben-Aroya, S. Jentsch, M.

51. T. Kubota, Y. Katou, R. Nakato, K. Shirahige, A. D. Donaldson, Replication-Coupled PCNA

54. I. Lacal, R. Ventura, T. J. Stevenson, A. J. Hannan, Epigenetic Inheritance: Concepts,

55. P. S. Kayne, U. J. Kim, M. Han, J. R. Mullen, F. Yoshizaki, M. Grunstein, Extremely conserved

57. J. E. Brownell, J. Zhou, T. Ranalli, “Tetrahymena Histone Acetyltransferase A: A Homolog to

58. F. W. Symington, J. Sprent, ; M Sarmiento, A. L. Glasebrook, F. W. Fitch, “RL72 (IgM antibodies

59. G. J. Hoppe, J. C. Tanny, A. D. Rudner, S. A. Gerber, S. Danaie, S. P. Gygi, D. Moazed, Steps in

61. L. N. Rusché, A. L. Kirchmaier, J. Rine, Ordered Nucleation and Spreading of Silenced

63. S. Imai, C. M. Armstrong, M. Kaeberlein, L. Guarente, Transcriptional silencing and longevity

66. A. A. Franco, W. M. Lam, P. M. Burgers, P. D. Kaufman, Histone deposition protein Asf1

67. V. K. Gali, D. Dickerson, Y. Katou, K. Fujiki, K. Shirahige, T. Owen-Hughes, T. Kubotaid, A. D.

70. C. M. Kondratick, J. M. Litman, K. v Shaffer, M. Todd Washington, L. M. Dieckman, Crystal