Cohesin Is Limiting for the Suppression of DNA Damage–
Induced Recombination between Homologous
Shay Covo, James W. Westmoreland, Dmitry A. Gordenin., Michael A. Resnick.*
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Research Triangle Park, North Carolina,
United States of America
Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is
generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be
reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes.
Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and
genome stability and that it suppresses DNA damage–induced interactions between homologues. We developed a gene
dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike
RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid
cohesion subunit genes affected survival of gamma-irradiated G2/M cells. The decreased survival reflected a reduction in
DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G2/M cells
with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The
increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage–induced
recombinants in G2/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome
integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced
recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in
cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.
Citation: Covo S, Westmoreland JW, Gordenin DA, Resnick MA (2010) Cohesin Is Limiting for the Suppression of DNA Damage–Induced Recombination between
Homologous Chromosomes. PLoS Genet 6(7): e1001006. doi:10.1371/journal.pgen.1001006
Editor: James E. Haber, Brandeis University, United States of America
Received February 2, 2010; Accepted May 27, 2010; Published July 1, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was supported by the Intramural Research Program of the NIEHS (project 065073). The funder had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Genome stability is maintained by a network of proteins that
ensure faithful DNA replication and efficient response to DNA
damage. Variation in levels of proteins across the cell cycle,
between tissues and even through natural fluctuations are common
[1,2,3] and could influence genome stability especially for proteins
that are present in limiting amounts. Proteins with limited
expression are likely to be weak links in genome maintenance
and, therefore, could be risk factors in disease, especially cancer
predisposition, when combined with environmental stress. This
could be particularly important for the cases where small,
environmentally relevant amounts of genotoxins inhibit a
mutation avoidance repair system . Even a cell with WT
genotype may be at risk for genome instability due to fluctuation in
expression of limiting proteins.
Many genes are involved in spontaneous and damage-induced
homologous recombination (HR) ensuring efficiency and accura-
cy. The repair of double-strand breaks (DSBs) by HR is an
evolutionarily conserved process (for review, see ) and is
generally considered error free since it uses information from an
undamaged DNA template. However, since HR can also occur
between related as well as identical sequences it can lead to
genomic instability through loss-of-heterozygosity (LOH) and
nonallelic recombination between repeats across the genome,
which can result in chromosome rearrangements [6,7]. These
changes are often detected in genetic disorders, cancer and during
evolution (discussed in, [8,9,10]).
Mutations in HR components can lead to genome instability and
cancer predisposition . Increased genome instability can also
result from changes in the amounts of wild type gene products
functioning in HR. In yeast, a genome wide analysis identified 178
genes with haplo-insufficiency causing increased chromosome loss
in the heterozygote state . Included was RAD55, which is
directly related to HR; it showed both chromosomal instability and
sensitivity to DNA damage when heterozygous. Haplo-insufficiency
for several human genes leads to DNA damage sensitivity, genome
instability and/or cancer susceptibility, suggesting they are present
in amounts that are limiting for HR [13,14].
We sought to identify more proteins that are present in limiting
amounts for HR-mediated DSB repair and to assess the
consequences of reduced levels. The identification of proteins that
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when limiting affect genome stability can be accomplished through
manipulation of gene dosage in polyploid cells. Small variations in
the amount of a protein can be accomplished with tetraploid
strains of the budding yeast Saccharomyces cerevisiae where gene
dosage can be varied over a factor of 4 from one (simplex) to four
copies (tetraplex; referred to as WT) by deleting copies of the gene
from homologous chromosomes. This scheme provides the
opportunity to address the relationship between gene dosage and
biological consequences for many genes. It also enables studies
reduced amounts of essential gene products. Importantly, unlike
other systems for down-regulating proteins, the amount of a
protein can be reduced without affecting the coding sequence or
other transcription/translation controls of the remaining alleles.
This approach was used for the yeast photolyase DNA repair gene
PHR1  which can reverse UV-induced pyrimidine dimers and
the RAD52 gene  which is essential for recombinational repair
of DSBs .
We applied the reduced gene dosage approach to three genes
that impact HR: RAD50, RAD51, and MCD1. The MRX complex
in yeast, which includes Rad50, is responsible for DSB recognition
and DNA resection, the first step in HR and in DNA damage
signaling at site-specific and in damage-induced genome wide
DSBs ([18,19] and references therein). The Rad51 protein which
is directly involved in recombination including homology search
and formation of joint molecule (for review see, ) was
previously suggested to be present in limiting amounts . We
found that changes in levels of Rad50 and Rad51 did not affect the
response to ionizing radiation.
We also investigated the consequences to genome stability of
reducing the dosage of genes affecting sister chromatid cohesion.
While not directly involved enzymatically in HR , the sister
chromatid cohesion complex (cohesin) that includes Mcd1, Smc3,
Smc1 and Irr1 is important in DSB repair in haploid yeast cells
( and for review, see ). Following induction of DSBs,
cohesin is recruited to DSBs via the DNA damage response
pathway [22,25]. The cohesin becomes cohesive even at
undamaged sites of the genome [26,27]. Although cohesin
facilitates DSB repair between sister chromatids, its impact when
homologous chromosomes are present is unknown. Recombina-
tion between sister chromatids is generally acknowledged to be
more efficient than between homologous chromosomes 
suggesting that cohesin inhibits recombination between homolo-
gous chromosomes. In this sense, cohesin might suppress
opportunities for LOH as well as nonallelic recombination and
chromosome rearrangements involving repeated DNAs. Previous-
ly it was shown that cohesin can influence the pattern of
recombination induced by a single DSB in a plasmid based assay
. However, since cohesin is an essential gene and viable
mutants are likely to be sensitive to ionizing radiation it is not
known what role it might play in maintaining recombination
fidelity when survival is high.
Here we show that even a modest reduction in the level of
cohesin dramatically increases the ability of c–radiation to induce
recombination between homologous chromosomes in the G2but
not the G1phase of the cell cycle even at low radiation doses when
survival is high. This finding, which also extends to UV-induced
recombination, suggests that cohesin confines recombinational
repair to sister chromatids even in the absence of DSBs, thereby
reducing the risk of genome instability.
Identifying limiting factors in DSB repair using a gene
In order to identify factors that are limiting for DSB repair,
pairs of tetraploid strains were produced that were simplex or WT
for genes of interest and examined for IR sensitivity. To develop
the simplex strains, diploids of opposite mating types were created
and transformed with gene inactivation cassettes containing
different antibiotic resistance markers as described in Figure 1.
The diploids were crossed to yield tetraploid strains with only two
functional copies (duplex). Tetraploids were confirmed by i) loss of
mating ability, ii) presence of resistance to G418 and hygromicin
antibiotics and iii) methionine prototrophy due to complementa-
tion of met2 and met6 mutants (see Materials and Methodss).
Finally, a simplex strain was created by inactivating one of the two
remaining functional genes in the duplex strain. Genotypes were
confirmed by PCR at all steps in construction.
Decrease in Mcd1, but not Rad50 or Rad51, enhances
sensitivity and reduces DSB repair
Changes in gene dosage of either RAD51 or RAD50 did not
affect gamma sensitivity (Figure 2) after exposure to 80 krad.
However, there was a marked increase in sensitivity for the MCD1
simplex compared to WT strains, which was not attributable to
growth effects (Figure 2 and Figure S1). In contrast, a temperature
sensitive mcd1-1 diploid cell shows high sensitivity to IR and slower
growth based on the appearance of small colonies after 2 days of
growth. As expected, reduction in the expression of each of the
respective proteins in the logarithmically growing simplex strains
was close to 4-fold (considering variability of Western blot
measurements) in comparison to WT as shown in Figure 2B.
Thus, it appears that unlike Rad50 and Rad51, the Mcd1 protein
is limiting for cellular responses to gamma radiation.
To address more precisely the importance of cohesin in cells
containing sister chromatids and to determine if there are subtle
effects in RAD50 and RAD51 simplex strains, cells were gamma
irradiated after nocadazole induced G2/M arrest. As shown in
Figure 3A, the MCD1 simplex strain was clearly more susceptible
to IR than WT. There was a 2-fold increase in the dose-modifying
factor (e.g., the same killing was achieved with half the dose) which
corresponds to a large difference in survival over the range of 20 to
80 krad (Figure 3A; 52% for the MCD1 simplex vs 87% for the
WT at 20 krad; p=0.009, n=13). While survival of the MCD1
The cellular concentrations of individual proteins are
expected to be kept within an optimal range, but protein
expression is often stochastic. Some proteins are known to
be in limiting amounts, so that even modest reduction can
lead to malfunction. Within the network of genes that
determine genome stability, proteins that are limiting
impose a risk for the cell, because fluctuation in their
amounts may start a cascade of genomic alternations that
will influence many biochemical pathways either under
normal growth conditions or in response to chromosome
damage. We sought to identify genes that are limiting for
DSB repair by lowering the dosage of key genes from 4 to
1 in tetraploid Saccharomyces cerevisiae strains. We found
that the complex that holds sister chromatid cohesion
together (cohesin) is limiting in DSB repair. In addition,
when it is reduced modestly, recombination between
homologous chromosomes is highly increased, suggesting
that the risk for loss of hetrozygosity (LOH) is increased
too. These results should also be considered in light of
increasing evidence that copy number variation can
impact cellular function.
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simplex strain was lower than WT, it could still tolerate many
DSBs (over 100, based on estimates from ). Since the genomes
of tetraploid yeast cells are somewhat unstable, we considered the
possibility that a portion of the MCD1 simplex population had
gained an extra copy of MCD1. To rule this out, we determined,
the survival of 10 MCD1 duplex and 19 MCD1 simplex isolates
after 80 krad exposure of cultures arrested by nocadazole. There
was no overlap between the duplex and the simplex cells. The
survivals of all the MCD1 simplex cultures were 3–300 fold less
than the median survival of the duplex MCD1 strains (data not
shown). Thus, if there are some cells in a simplex population that
have an additional copy of MCD1, their frequency is small and
would only be expected to result in an underestimation of the
induced recombination frequencies (see results and discussion
The role of Mcd1 in resistance to IR was also confirmed with
homozygous diploids carrying the mcd1-1 temperature-sensitive
allele when cells were plated at the semi-permissive temperature
32uC after irradiation (Figure S2). Neither RAD50 nor RAD51
simplex strain showed any change from WT strain in the dose
strain were somewhat more sensitive to IR at high doses.
Based on these results, we chose to focus the rest of this study
A reduced level of Mcd1 does not affect radiation
sensitivity of G1 cells, but does affect sensitivity and DSB
repair in G2/M cells
Since sister chromatid cohesion is established during S phase
and disrupted during anaphase we asked whether cohesin affects
the response to IR during the G1stage of the cell cycle, when cells
lack sister chromatids. Previous studies with yeast have been
restricted to survival or DSB repair measurements with haploid
cells, which would lack any opportunity for repair between
homologous chromosomes. The absence of repair of radiation-
induced DSBs by nonhomologous end-joining , unlike
mammalian cells, render yeast a good model for addressing
defects in homologous recombination. The MCD1 simplex and
WT cells were grown for 3 days to stationary phase (.90% G1
cells, based on cell morphology) and exposed to IR. No significant
difference was observed between WT and MCD1 simplex cells at
this stage. Importantly, the response of MCD1 simplex cells
irradiated at G2/M or as stationary cells (primarily G1), was
comparable to that of WT cells in stationary cells (Figure 3). These
results suggest that the cohesin function associated with sister
chromatids has little role in DSB repair that might occur between
homologous chromosomes in G1cells.
We examined directly the impact of decreased levels of Mcd1
on DSB repair in the G2/M cells using pulsed field gel
electrophoresis (PFGE) [6,18]. PFGE separates individual chro-
mosomes on the basis of size so that gamma induced DSBs and
repair can be readily assessed (Figure 4). The efficiency of DSB
repair is determined by an analysis of restitution of full size
chromosomes during post-irradiation incubation (see Materials
and Methods). While repair was detected, the MCD1 simplex
strain clearly exhibited reduced repair capacity in comparison to
WT cells as shown for cells irradiated with 80 krad, corresponding
to ,600 DSBs/cell (Figure 4), [6,18]. The reduced levels of Mcd1
significantly affected the rate of repair at 1 to 4 hr post-irradiation
incubation (see Figure 4). For example, within 1 hr after IR the
WT cells repaired ,70% of the DSBs induced by 80 krad while
half as many were repaired in the MCD1 simplex strain (Figure 4B).
Increasing post-irradiation incubation time to 4 hr led to more
repair in the WT and MCD1 simplex cells; however, there were
still about 4 times more unrepaired breaks in the MCD1 simplex
than the WT cells (23% vs 6%). At a lower dose (40 krad; Figure 4),
reduced levels of Mcd1 had less of an impact consistent with the
smaller differences in killing (Figure 3A). We note the limited
ability of the PFGE repair assay to detect small differences in
DSBR capacity. This is relevant to considerations of IR induced
lethality since unrepaired DSBs appear to have a dominant effect
on cell killing .
Thus, we establish that the level of Mcd1 is critical both for
efficient repair of DSBs and maintaining resistance to radiation in
Figure 1. Development of tetraploid simplex strains. Within
Diploids 1 and 2, which have opposite mating types, one copy of the
gene of interest (MFG 1, i.e., my favorite gene) was inactivated in each
diploid by transformation with kanamycin or hygromicin cassettes
that target deletions. The resulting diploids were crossed to create
Met+(see Materials and Methods) KanRand HygRtetraploids with
two copies (duplex) of MFG1. Finally, a third copy of MFG1 was
inactivated by transforming the duplex strain with a URA3 targeting
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Reduction in cohesin subunits Mcd1 and Smc3 increases
IR–induced recombination between homologous
chromosomes in G2 cells
The decreased DSB repair in MCD1 simplex cells arrested at G2/
M suggests that there might be a change in interactions between
homologous chromosomes. To address directly recombination
between homologous chromosomes, we developed the genetic
reporter described in Figure 5. The tetraploid cells carry two
versions of chromosome II where two of the four chromosomes
contain the 59 portion of the TYR1 and the other two carry the 39
portion. The 39 and 59 truncations have a 400 bp overlap; such that
homologous recombination can lead to Tyr+cells (see Figure 5 and
Materials and Methods). The TYR1 recombinants are likely to arise
through a gene conversion process that covers only one of the alleles
either associated or not associated with cross-over (Figure 5A). They
could also occur by a DSB in the homologous region between the
two heteroalleles generating a tract that ends between the alleles,
which could result in a reciprocal exchange (Figure 5). We assume
that changes in the frequency of Tyr+recombinants are directly
correlated with changes in number of interactions between
We found that the spontaneous rates of Tyr+recombination
were not affected by the level of Mcd1. The median rates for the
simplex and the WT MCD1 strains were 2.561026(1.1–
2.961026; 95% confidence interval) and 1.561026(1–261026;
95% confidence interval). Exposure to IR increased the frequen-
cies of Tyr+recombinants in G2/M arrested cells in all strains
examined. The efficiency of induction in MCD1 simplex cells (,5–
1061026recombinants/survivor/krad) was approximately 10-fold
greater than in WT cells over a range extending from sublethal
doses to ,50% survival at 20 krad (Figure 6A; see Figure 3A for
survival), even though DSBR is efficient (Figure 4). (Based on an
induction efficiency of 0.07 DSB/mb/krad  there are sufficient
DSBs to account for the observed recombinants even if all events
are generated by DSBs in the 400 nt overlap region.) Since the
recombination assay scores infrequent events (,0.1% of the
population), it is possible that some of the Tyr+colonies were not
MCD1 simplex. In order to estimate a change in mcd1 deletion
alleles, 160 presumptive MCD1 simplex Tyr+colonies arising after
nocodazole arrest and 20 krad treatment, were replica-plated to
the appropriate media to verify the presence of the simplex
markers (G418 and Hygromycin resistance and Ura+phenotype).
Only 4 colonies lost one of these markers (2.5%), suggesting that
most of the colonies were actually MCD1 simplex.
The MCD1 duplex cells (two functional gene copies out of 4, see
Figure 1) also showed a significant elevation in IR-induced
recombination frequency (Figure 6B). For example, for WT and
MCD1 duplex cells irradiated with 10 krad the induced frequen-
cies were 106361026and 286361026, respectively (p=0.013,
n=9). Responses were very different with G1 stationary cells
where HR interactions are restricted to homologous chromo-
somes. The induction of recombination in the stationary cells was
marginally influenced by MCD1 gene dosage (,2 fold; Figure 6A).
Interestingly, the recombination frequencies in the WT cells
irradiated at stationary stage matched the HR response of the
simplex strain irradiated at G2/M (Figure 6A).
The cohesin complex itself appears to be limiting since simplex
strains of SMC3, another member of the complex, also showed
elevated IR-induced HR between homologues in G2/M cells
(Figure 6A). At 10 and 20 krad, corresponding to 80% and 70%
survival, respectively, the Tyr+recombinant frequency in the
SMC3 simplex strain was about 5-fold higher than the WT.
Figure 2. Cells that are simplex for MCD1, but not RAD50 or RAD51, are sensitive to ionizing radiation. (A) Late logarithmically growing
cells (56107cells/ml) of tetraploid strains with 4 copies (WT) or one copy (simplex) of MCD1, RAD50 and RAD51 as well as mcd1-1 diploid cells were
spotted on YPDA plates. The control and irradiated plates (80 krad) were incubated for two days at 30uC. (B) Logarithmically growing cultures of WT,
and simplex strains (presented as ‘‘S’’) for MCD1, RAD50 and RAD51 were harvested in SDS PAGE sample buffer and separated on SDS PAGE. Protein
amounts were determined by western blot analysis using antibodies; histone H3 (presented as ‘‘H3’’) was used as a loading control. The normalized
intensity of each protein in the simplex strain was divided by the normalized intensity in WT to give the simplex/WT ratio.
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The impact that a reduced level of Mcd1 has on recombination
between homologues was not specific to tetraploid cells. We also
addressed the consequences of lowering Mcd1 function in a
diploid strain. As shown in Figure S3, the levels of IR-induced
recombination in G2/M arrested WT tetraploid is not higher than
in diploid cells, suggesting that the additional chromosomes alone
do not increase opportunities for recombination. Since the
temperature sensitive mcd1-1 mutation has frequently been
employed to address the role of cohesin in haploid cells , we
also investigated IR-induced recombination in a homozygous
mcd1-1 diploid strain at the semi-permissive temperature of 32uC.
The level of induced TYR1 recombination was 3-fold higher
(186361026vs 6061061026; p-value 0.002, n=6) than in the
WT diploid following exposure to 20 krad (there was a 5-fold
difference in survival; see Figure S2). However, the impact on
recombination was less than for the MCD1 simplex strain
(discussed below). Since the MCD1 duplex showed higher
recombination frequencies than the WT tetraploid (Figure 6B), it
was expected that an MCD1 hetrozygous diploid would have an
elevated frequency of induced recombination between homolo-
gous chromosomes. Indeed, the induced frequency for the MCD1
hetrozygote was slightly higher than for the WT diploid following
exposure to 20 krad: 286361026and 186361026, respectively.
A similar difference was observed at 40 krad (43 vs 28
recombinants/1026survivors, respectively). For both doses the
differences were statistically significant based on a one-tailed t test
(p=0.0194 and 0.0273 for 20 and 40 krad, respectively; n=10).
The differences between homozygous and heterozygous MCD1
diploids are smaller than the differences between WT tetraploid
and MCD1 duplex, suggesting that there is an additional
component(s) that further sensitizes tetraploid cells to cohesion
defects. In support of this view, mcd1-1 tetraploid cells are inviable
even at a temperature that enabled growth of the corresponding
In summary, the MCD1 simplex strain shows lower global DSB
repair capacity but higher radiation-induced recombination
frequencies between homologous chromosomes than the WT
cells. We conclude that the limiting levels of cohesin are sufficient
to direct repair of gamma induced DSBs towards sister chromatids
and that reductions in cohesin open opportunities for recombina-
tion between homologous chromosomes.
Decreased MCD1 gene dosage also increases UV– and
UV radiation can induce recombination between sister
chromatids and homologous chromosomes . It does not
generate DSBs directly, although they might arise through repair
of closely spaced lesions on complementary strands or during
Figure 3. MCD1 simplex cells are more sensitive than the WT to IR as G2/M but not G1(stationary) phase cells. (A) Nocodazole-arrested
WT RAD50, RAD51 and MCD1 simplex cells (G2/M) or (B) WT and MCD1cells from 3 day stationary cultures (G1) were irradiated with the indicated
doses. Cells were spread on YPDA (described in Materials and Methods under ‘‘Nocodazole arrest, gamma irradiation, and post irradiation
incubation’’) or complete synthetic media plates (there was no difference in survival between the two types of media). Survival was determined after
2–3 days. Survival was determined from at least 6 cultures for each genotype; error bars correspond to the standard error (SEM).
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replication. Surprisingly, the reduction in MCD1 gene dosage
resulted in UV-induced increases in HR frequencies in G2/M cells
comparable to those for IR (Figure 6A and Figure 6C): 2063/106
survivors for the simplex strain vs 260.7/106survivors for WT at
10 J/m2(p=0.0001). The difference was increased to 20-fold,
reaching 132638 recombinants/106survivors at 40 J/m2vs 862
recombinants/106for WT cells (Figure 6C).
Based on experiments with rad52 haploid cells that are unable to
repair DSBs, the differences between the MCD1 simplex and WT
is not attributable to UV being able to generate DSBs directly or
indirectly in the G2/M cells. While survival after 40 J/m2UV
irradiation of rad52 cells was 15%, survival after 20 krad was less
than 0.1% indicating that many more DSBs occur when cells are
irradiated with 20 krad than 40 J/m2UV. The recombination is
likely to arise in the G2/M cells rather than in the subsequent S
phase. UV- induced recombination in WT stationary cells was
over 10-fold greater than in G2/M (Figure 6C) suggesting that UV
lesions generated at G1 or entering the next S phase are still highly
recombinogenic even in WT cells. Surprisingly, the recombination
frequency for UV-irradiated stationary phase MCD1 simplex cells
was only 2-fold higher than for WT cells (Figure 6C), far less of an
effect than for cells irradiated at G2/M.
The low UV-induced recombination rates for WT cells
irradiated at G2/M could stem from very efficient nucleotide
excision repair that removes UV lesions at G2, possibly suggesting
that the high recombination rates of MCD1 simplex might be due
to reduced efficiency in removal UV lesions. However, the MCD1
simplex strain was not sensitive to UV. Survival following exposure
to 10 and 20 J/m2at G2/M was 100% for both the WT and
MCD1 simplex strains; even at 40 J/m2the survival was similar for
Figure 4. DSBs are repaired slowly in MCD1 simplex as compared to WT cells. Logarithmically growing MCD1 simplex and WT strains in
YPDA medium at 30uC were arrested in nocodazole for 2.5 hours and irradiated with the indicated dose (See Materials and Methods). Samples were
taken before and immediately after radiation. Cells were re-suspended in warm YPDA medium containing nocadazole and incubation was continued.
Samples were taken at 1 and 4 hr. (A) The chromosomal DNA was displayed using PFGE. (B) The induction of DSBs and subsequent repair was
calculated from the intensity of the retained chromosomes. A detailed description of the preparation of DNA plugs, PFGE conditions and DSB
quantification is presented in Materials and Methods.
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the MCD1 simplex and WT strains (68620% and 89613%,
respectively). Also, irradiation of unsynchronized cells showed no
difference between WT and MCD1 simplex cells even at high UV
doses (Figure S4).
The DNA synthesis inhibitor hydroxyurea (HU) can generate
stalled replication forks leading to DSBs that can be rescued by
recombination . We asked whether differences in MCD1 levels
could influence HU-induced recombination between homologous
Figure 5. Genetic reporter for recombination between heteroalles residing on homologous chromosomes in tetraploid strains. (A)
The tetraploid cells carry two versions of chromosome II with deletions within the TYR1 ORF: the ‘‘ty’’-allele contains a 59 portion of the TYR1 ORF
(nucleotides 1–700) and the ‘‘yr1’’-allele contains a 39 portion (nucleotides 300–1358). The green box represents the ORF of the TYR1 gene; the striped
boxes represent the missing DNA sequences of the mutants; and the light blue rectangle represents the area of homology between the
chromosomes. Note: for simplicity only one of each pair of sister chromatids of the G2/M cells are presented (thereby, having the appearance of G1
cells). (For a complete description of recombination and segregation at mitosis see Figure S7.) Gene conversion with or without crossing-over as well
as reciprocal exchange between heteroalleles can generate Tyr+cells. Recombination leading to Tyr+can generate different combinations of TYR1
alleles within the resulting tetraploid cell. A large conversion tract will result in 3 types of alleles regardless of associated crossing-over: the original
truncated parental heteroalleles and the TYR1+converted allele. A reciprocal exchange that occurs at short conversion region will yield a forth allele
‘‘y’’ retains only a small portion of the gene. Depending on segregation of sister chromatids at mitosis, half the Tyr+recombinants that arise by a
reciprocal exchange would not possess the ‘‘y’’ allele (for details see Figure S7). (B) The 4 alleles described in (A) can be distinguished by size of PCR
products using the following primers: 59GAATACCGTAGCACTTGAAGGAAAGAGGACAGCATATCCA 59CACAAAAGAAGGCCTAATATTATAGGAAATCAG-
CATTAAAAAC. The allele sizes are 1360 bp (TYR1), 1060 bp (‘‘yr1’’), 700 bp (‘‘ty’’) and 400 bp (‘‘y’’). Presented are PCR products of the TYR1 locus from
4 colonies obtained after UV irradiation (40 J/m2) of the MCD1 simplex strains. Tyr+colonies that result from a reciprocal exchange event can be
identified by the presence of a ‘‘y’’ allele (encircled). The ‘‘M’’ corresponds to DNA molecular size markers.
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Figure 6. Cohesin is limiting in the suppression of damage-induced recombination. (A) Induction of recombination by ionizing radiation of
nocodozole-arrested WT, MCD1 and SMC3 simplex cells and stationary WT and MCD1 simplex cells. After irradiation, cells were plated to synthetic
complete (SC) or SC lacking tyrosine (SC-Tyr) and incubated for 2–3 days. Shown are the net recombination frequencies (induced minus ‘‘no
irradiation’’). (B) IR-induced recombination in nocodazole arrested MCD1 duplex cells (as a comparison, the WT data was pooled from panel A and
four more WT cultures that were done side-by-side). (C) UV-induction of recombination in nocodozole-arrested and stationary WT and MCD1 simplex
cells. (D) Induction of recombination by hydroxyurea. Logarithmically growing WT or MCD1 simplex cells were treated with HU overnight. Cells were
then spread on complete and Tyr2plates. Presented are induced recombination frequencies (the frequency measured after HU treatment minus the
frequency measured without treatment; see legend to Figure S5 for a detailed description). Recombination frequencies were obtained from at least 8
cultures for each genotype and for each DNA damaging agent.
Reduced Cohesin Enhances Induced Recombination
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chromosomes. Logarithmically growing cells were treated with
HU overnight and recombination between homologous chromo-
somes was determined. Growth inhibition ranged between 25%
and 90% (50 to 150 mM) and was somewhat higher in the simplex
as compared to the WT strain (Figure S5). At these doses there was
induction of recombinants in both the MCD1 simplex and WT
strains (Figure 6D); however, the frequencies (,20 recombinants/
106survivors) were much lower than for IR- and UV-induced
recombination (Figure 6). Reduction in the level of Mcd1 resulted
in only a small (,2-fold) increase in HU-induced recombination
over the WT strain.
Reduced Mcd1 levels open the genome to gene
conversion and crossing over between homologous
Since gene conversion may be associated with crossing-over at
the chromosome level, increases in Tyr+prototrophs - are likely to
reflect increases in cross-overs and, therefore, LOH. If reciprocal
crossing-over occurs in the G2stage of the cell cycle, half the
events would result in chromosomes with long stretches of LOH,
depending on segregation of the sister chromatids while crossing-
over in G1would not yield LOH (see Figure S6).
Reciprocal exchange (RE) products containing the ‘‘y’’ allele
result from cross-overs that fall between the two tyr1 heteroalleles
(Figure 5A). These can be identified by PCR genotyping
(Figure 5B) as short fragments distinct from the wild type
recombinant allele (TYR1) and the alleles that were unaffected
by the recombination event (‘‘tyr’’ and ‘‘yr1’’). If a crossing-over
event leading to Tyr+occurs in G1 or in S phase cells prior to
replication of the TYR1 region, the ‘‘y’’ allele would appear in all
progeny cells. However, for cross-overs in G2/M only half the ‘‘y’’
alleles would be recovered because of sister chromatid segregation
(see Figure S7); therefore, the observed frequency of ‘‘y’’ alleles
among the Tyr+recombinants is a minimal estimate of the actual
RE frequency. We note that the above PCR based assay only
detects cross-overs with a short conversion tract, while events with
a long tract will not be discernable (Figure 5) (see also  for
conversion tract length in mitotic crossing over).
We determined the cross-overs among the Tyr+recombinants
after exposure of G2/M cells to 20 krad IR or 40 J/m2. The ‘‘y’’
allele was observed in a small fraction of the Tyr+recombinants
appearing after IR and UV exposure of the WT and simplex
strains (Figure 7A). The minimum frequency of REs among Tyr+
recombinants did not differ significantly between the MCD1
simplex and WT Tyr+strains. Based on the overall recombination
frequencies presented in Figure 6A and Figure 6C and assuming
recombination occurred in the G2/M cells, the expected induced
RE frequency in MCD1simplex is much higher than in WT cells,
as described in Figure 7B. Thus, while a reduction in the amount
of Mcd1 does not change the recombination fate in terms of cross-
overs vs no cross-overs, we suggest that the a reduced level of
Mcd1 places the genome at considerable risk for both IR and UV
induced gene conversion and crossing-over.
The gene dosage approach to identifying limiting factors
in genome stability
In order to address the consequences of moderate changes in
key proteins responsible for DSB repair we developed tetraploid
strains with changes in dosage of the corresponding genes. Using
survival response to DNA damage as a screening tool, Mcd1 was
identified as a limiting factor in DSBR unlike Rad50 and Rad51
whose complete elimination confers extreme sensitivity to IR. This
approach by itself may provide tools for identifying targets that
could be used for radiotherapy sensitization (see below). More
importantly, this approach allowed us to focus on cohesin as a
limiting factor in maintaining genome stability. We note that other
proteins are also limiting for genome stability maintenance in yeast
and mammalian cells as demonstrated for several genes that
exhibit haplo-insufficiency [12,13]. We were able to show that
recombination between homologous chromosome is highly
increased in a cohesin simplex strain (Figure 6) suggesting that
cohesin channels DSB repair to sister chromatids and suppresses
recombination between homologous chromosomes (Figure 8).
As illustrated in Figure S6 restricting recombinational repair to
sister chromatids reduces the likelihood of LOHas well as nonallelic
recombination, thereby decreasing opportunities for damage-
induced variations in genomic structure . The combination of
LOH and nonallelic recombination can be a powerful source of
carcinogenesis. While it is well established that cohesin facilitates
DSB recombinational repair through stabilization of sister chroma-
tidinteractions,suggestionsthat thereisa correspondingdecrease in
opportunities for DSB repair through homologous recombination
have lacked experimental support, especially since experiments
were done in haploid yeast. Furthermore, there has been no
discussion of a potential impact on recombination induced by other
agents, particularly those that do not generate DSBs. We have
demonstrated a dramatic (nearly 10-fold) IR-induced increase in
recombinationbetween homologueseven atlow, sublethal doses(5–
10 krad, Figure 6A and Figure 3A) under conditions of moderately
reduced levels of cohesin and normal mechanisms of cellular
expression. Previous experiments have utilized temperature sensi-
tive cohesin mutants of the essential MCD1 gene,which grow poorly
and are radiation sensitive at semi-permissive temperatures
(Figure 2, Figure S2). The recombination frequencies of the mcd1-
1 strain are also greater than for WT. However, the recombination
frequency for mcd1-1 at 20 krad (20% survival) is comparable (60/
106survivors) to that estimated for the simplex irradiated with one
third the dose (Figure 6A), corresponding to 100% survival
(Figure 3A). Also, the MCD1 simplex strain had a growth rate
comparable to WT (Figure S1). Taken together, we suggest that the
amount of cohesin is limiting for suppression of recombination
between homologous chromosomes. We speculate that there is
enough cohesin to hold the sister chromatids after DNA replication,
but the non-cohesive reservoir of cohesin in the simplex strain is
limiting such that it cannot suppress DNA damage-induced events
that occur at G2.
The biological importance of a 3-fold reduction in the amount
of a protein (Figure 2B) leading to a 10-fold increase in
recombination frequency may lie in the stochastic pattern of
protein expression where 2–3 fold changes in the amount of
proteins appear to be relatively common . For most proteins
which are not limiting, such changes are not expected to affect the
biological outcome but here we show that a small perturbation in
cohesin may place a cell at risk for genome instability.
The reduction in gene dosage and protein levels in RAD50 or
RAD51 simplexes strains did not lead to IR sensitivity. The lack of
difference in sensitivity for RAD51 may be related to the much
larger number of molecules per cell: 7000 molecules of Rad51 vs
1000 Mcd1 in logarithmically growing haploid cells . However,
expectations based strictly on number of molecules present must
be balanced against number of molecules needed for function. For
example, the Rad51 repair unit is a multiprotein, single-stranded
DNA filament that is likely restricted to regions of DNA
undergoing repair. Similarly, even though the number of Rad50
molecules is comparable to that for Mcd1 (,800 per cell,
)restricting these molecules to sites of damage may limit the
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need for Rad50. Interestingly, deletion of the RAD50 gene results
in hyper-recombination between homologous chromosomes 
which can be explained by reduced recruitment of cohesin to
DSBs . The RAD50 simplex did not exhibit hyper-recombi-
nation frequencies (data not shown), suggesting there is enough
Rad50 also for cohesin recruitment. The cellular requirements for
cohesin are likely much larger given that these molecules are
utilized in sister chromatids across the genome. The mean distance
between cohesin binding sites is 11 kb [37,38] corresponding to
around 1000 binding sites in the genome. Also, large amounts of
cohesin are recruited directly to DSBs following DNA damage
 and ‘‘noncohesive’’ cohesin complexes become ‘‘cohesive’’ at
sites distal to DSBs [26,27]. Limiting amounts of cohesin raises the
question of why not more. Possibly, too much cohesin may
increase the risk of nondisjunction at mitosis, a view that is
supported by the antiestablishment activity of the Rad61-Pds5-
Scc3 complex towards cohesin in G2[39,40].
While there have been suggestions that tetraploid and diploid
yeast may differ in ability to maintain their genomes , the
survival responses to IR are comparable on a per lesion basis from
diploids to tetraploid cells (summarized in ). Furthermore, we
found that IR-induced recombination between homologous
chromosomes of G2/M arrested diploid and tetraploid cells did
not differ significantly (Figure S3).
A general role for Mcd1 in confining recombination to
We found that Mcd1 suppresses UV- as well as IR-induced
recombination between homologous chromosomes. Surprisingly,
the UV-induced frequencies in G2/M cells were increased nearly
Figure 7. Both reciprocal and non-reciprocal exchanges are found in DNA damage–induced TYR1 recombinants. (A) Minimal
estimation of reciprocal exchanges (RE) was obtained by PCR amplification of TYR1 locus of Tyr+colonies (details in text and in Figure 5) (The
numbers of events were ‘‘y’’ was identified or not are presented). (B) Calculation of excepted frequency of RE in TYR1 locus of irradiated cells. The
minimal estimate for RE frequency among Tyr+recombinants (7A) was multiplied by 2, assuming that all events occurred in G2(Figure S7) and then
was multiplied by the frequency of Tyr+induced recombinants as measured in Figure 6.
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20-fold in simplex MCD1 cells as compared to WT cells
(Figure 6C). For UV, the increased recombination is unlikely to
be related to DSBs. First, the recombination frequency is similar
for 40 J/m2UV and 20 krad IR. At this dose of ionizing radiation,
DSBs are readily detected, while UV-induced DSBs would be
rare. Second, Lettier et al.  observed that most UV-induced
recombination events are independent of DSB repair since they
occur in a rad52 mutant that is completely lacking in DSB repair.
Until now, the link between cohesin and homologous recombina-
tion was strictly based on the relationship between DSB induction
and recruitment of cohesin to the break site. From our results we
conclude that cohesin restricts potential recombinational interac-
tions induced by ionizing and UV recombination to sister
chromatids (Figure 6). We consider it likely that recombination
induced by other agents would be similarly affected. Cohesin may
accomplish sister chromatid preference simply by holding
chromatids in close vicinity at normal cohesion attachment sites
[37,38] such that the undamaged sister becomes the preferred
recombination partner. In addition, cohesin may channel
recombination to sister chromatids because it is recruited directly
to DSBs. Exposure of single strand DNA at a DSB was shown to
be important for recruiting cohesin; however, single strand DNA
intermediates are found in other DNA repair pathways including
repair of UV lesions. In addition, rare DSBs associated with UV
damage might lead to greater amounts of cohesion between sister
chromatids across the genome as demonstrated for a site-specific
single DSB [26,27].
The effects of HU and the role of MCD1 gene dosage on
recombination differed considerably from IR and UV. HU-
induced recombination was only marginally elevated in the MCD1
simplex compared to WT (Figure 6D) strain. While HU can cause
fork collapse, it might be counteracted by a back-up mechanism(s)
that would also be anti-recombinogenic, for example, by Srs2
helicase recruitment via PCNA sumoylation .
Implications for limited cohesin complex and restriction
of recombination to sister chromatids
The cohesin complex and its functions are evolutionarily
conserved across eukaryotes (for review see [24,44] and references
within). Mammalian cohesin is recruited to DSBs and is part of the
ATM signal transduction and important for survival after IR.
[45,46]. In addition, the Smc3 cohesin subunit is acetylated to
establish cohesin, both in yeast and human cells [47,48].
Therefore, cohesin function in DSBR is probably conserved. It
will be interesting to determine if cohesin is limiting for responses
to DSB inducing agents in mammalian cells. If this is the case, then
cohesin might be a useful target during cancer treatment for
sensitizing cells to radiation and other drugs that break DNA.
Unlike fully differentiated cells, cancer cells spend more time in G2
and S phase, when recombination is highly efficient. Targeting
cohesin might be especially efficient when combined with cell cycle
inhibitors that cause G2arrest. It is interesting that mutations in
cohesin and related genes were found in many cancer cells that
show chromosome instability (CIN). In addition, reduction of the
amount of cohesin using RNAi leads to a CIN phenotype in cells
with a near diploid genome. Included among the CIN events were
the development of tetraploid genomes ; hence, a primary
defect in cohesin may generate tetraploid cells with further defects
in cohesion and genome stability.
While the present results indicate a general role of cohesin in
control of HR, our overall approach can provide useful insights
into genome dynamics as well as genetic processes associated with
tetraploidy. Tetraploid cells are common among eukaryotes and
during evolution  and show unique characteristics regarding
chromosome dynamics. In yeast, polyploid cells exhibit increased
genome instability in comparison to diploids  which makes
them an interesting model for a complex genome. Importantly,
mammalian hepatocytes, frequently give rise to polyploids . It
is worth noting that hepatocytes are continuously exposed to
genotoxic insults and polyploidy is often associated with the
carcinogenesis process . Therefore, the damage-inducible
increase in recombination observed in MCD1 simplex cells might
result in further genome instability in natural or transformed
Finally, we describe here an experimental design that can be
used to search for subtle changes in essential and nonessential
factors that are limiting for genome stability. Reduction in these
factors can synergize with modest (i.e., high survival) levels of
genotoxic stress to dramatically increase genetic change. Impor-
tantly, our approach utilizes normal, wild type proteins and native
gene expression regulation thereby eliminating the uncertainty
associated with mutations and variations in gene expression.
Tetraploids provide a wider opportunity to vary gene dosage as
compared to the simple homozygote-heterozygote approach in
diploids and may be more suited for addressing implications of
copy number variation, as found in the human genome .
Materials and Methods
For a list of strains, see Table 1 below. Each simplex strain
described in the table represents the genotype of at least 2 more
independent isolates that originated from 2 independent duplex
parents. Haploid strains were derivatives of E134  and its met2
and met6 derivatives DAG 647 and DAG 645 respectively. ura 3–
52 has been replaced by a complete deletion that gave rise to
strains CS1004 and CS1006 (see below). CS1004 (relevant
genotype MATa met 6-DEL) was transformed with CORE cassette
 targeted to the 39 end of TYR1, starting at nucleotide 700 of
the ORF. Briefly, the following primers were used to amplify
G418Rin tandem with the URA3 cassette from pCORE  in
order to create a 59 tyr1 allele.
TCGACACTGG and 59TTATGTATTTCTTTTTTCAGCG-
T-TCCTTACCATTAAGTTGATC. In parallel the CS1006
(relevant genotype MATa met 2-DEL) was transformed with a
CORE cassette targeted to the 59 part of the TYR1 (nucleotide #1
at ORF) in order to create a 39 tyr1 allele. This was done by
Figure 8. Cohesin channels HR to sister chromatids. Presented is
a model in which cohesin-mediated interactions between sister
chromatids reduces opportunities for damage-induced recombination
between homologous chromosomes.
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amplifying CORE cassette with primers 59ATGGTATCAGAG-
GTATAATTGGT-GAGCTCGTTTTCGACACTGG and 59T-
After selection of the CORE integrants, the CORE was
removed  from CS1004 (met6-DEL) using oligonucleotides
ACCGTTCGGCCGCTGAAA and 59TTTCAGCGGCCGAA-
The CORE was removed from CS1006 derivative (met2-DEL)
by introducing a portion of the TYR1 gene PCR fragment defined
by primers 59TGAAGGAAAGAGGACAGCATATCCACTT-
TACCGTGCATTCCCTTCATG and 59GCCACTTGTTCG-
Loss of CORE was identified by conversion of strain to G418
sensitive and 5FOA resistant. At the end of this stage, two sets of
strains were created; CS1004 derivative with met6 DEL and with
tyr1 allele of 1–700 bp and CS1006 derivative with tyr1 allele 300–
Diploid cells were made from derivatives of the above (Strains
CS 1061 and CS 1064 see Table 1) by introducing plasmid (YEp-
HO) encoding HO endonuclease under its native promoter to the
haploid strains. Non-mating cells were identified as potential
diploids and confirmed by ability to sporulate. Each a/a diploid
was then transformed with a pGal-HOT plasmid were HO is
inducible by galactose . Cells were grown 6 hr on galactose
containing media to induce mating type switching. MATaa and aa
cells were identified by mating test. The aa and aa strains were
isolated from each set of haploid strains (met 6-DEL 59 tyr1 allele
and met 2-DEL 39 tyr1 allele). The MATaa and aa cells served as
Diploid 1and 2 as shown in Figure 1.
WT tetraploid cells were obtained by crossing several diploid
isolates with opposite mating strains and complementing met
mutations. Cells were selected on media lacking methionine and
confirmed to be non-mating. They also exhibited spontaneous and
UV induced Tyr+ recombination.
MCD1 simplex strain formation
The following oligonucleotides were used to create G418Rand
HygromicinRcassettes that could be targeted to MCD1 open
Table 1. Intermediate steps in strain construction and final strains.
CS 1004 Starting haploid type 1 met6-DEL MATa ade5-1 his7-2 leu2-3,112 trp1-289 ura3-Del met6-DEL
CS 1006Starting haploid type 2 met2-DEL MATa ade5-1 his7-2 leu2-3,112 trp1-289 ura3-Del met2-DEL
CS 106139 tyr1 alleleAs CS 1006 with tyr1 300–1359 trp1-DEL
CS 106459 tyr1 allele As CS 1004 with tyr1 1–700 trp1-DEL
CS 1120 mcd1-1 As CS 1061 but mcd1-1
CS 1122mcd1-1 As CS 1064 but mcd1-1
CS 2050 (Diploid)As CS 1061 but MATaa
CS 2052 (Diploid)As CS 1064 but MATaa
CS 2064 Starting diploid 1 (Figure 1)As CS 2050 but MATaa
CS 2065Starting diploid 2 (Figure 1) As CS 2052 but MATaa
CS 2200MCD1 hetrozygote (Diploid)As CS2064 but MCD1/mcd1::KAN
CS 2222MCD1 hetrozygote (Diploid)As CS2065 but MCD1/mcd1::Hygromycin
CS 2274SMC3 heterozygote (Diploid)As CS2064 but SMC3/smc3::KAN
CS 2277SMC3 heterozygote (Diploid) As CS2065 but SMC3/smc3::Hygromycin
CS 2107RAD50 heterozygote (Diploid) As CS2065 but RAD50/rad50::KAN
CS 2208RAD50 heterozygote (Diploid) As CS2064 but RAD50/rad50::Hygromycin
CS 2251 Rad51 heterozygote (Diploid) As CS2064 but RAD51/rad50::Hygromycin
CS 2255 rad51 homozygote (Diploid) As CS2065 but rad51::KAN/rad51::URA3
CS 2054WT (Diploid)Cross CS1061XCS1064
CS 2259mcd1-1 (Diploid)Cross CS1120XCS1122
CS 4021WT tetraplex (Tetraploid) Cross of CS2064XCS2065
CS 4175MCD1 duplex (Tetraploid)MCD1/MCD1/mcd1:: Hygromycin/mcd1::KAN (Cross of CS2222XCS2200)
CS 4229SMC3 duplex (Tetraploid) SMC3/SMC3/smc3:: Hygromycin/smc3::KAN (Cross of CS2274XCS2276)
CS 4143RAD50 duplex (Tetraploid)RAD50/RAD50/rad50:: Hygromycin/rad50::KAN Cross of (CS2107XCS2208)
CS 4207MCD1 simplex (Tetraploid) MCD1/mcd1::URA3/mcd1::Hygromycin/mcd1::KAN (based on CS 4175)
CS 4238SMC3 simplex (Tetraploid)SMC3/smc3:URA3/smc3:: Hygromycin/smc3::KAN (based on CS 4229)
CS 4157 RAD50 simplex (Tetraploid)RAD50/rad50::URA3/rad50:: Hygromycin/rad50::KAN (based on CS 4143)
CS 4240RAD51 simplex (Tetraploid)Cross CS2251XCS2255
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reading frame using plasmids pFA6 and pAG32 respectively:
TACGCTGCAGGTCGACGGATCCCC and 59TTAAAGTC-
The MCD1 heterozygote diploid was created by transforming
Diploid 1 and 2 cells with the targeted G418Rand HygromicinR
cassettes, respectively. Independent MCD1 heterozygote isolates
derived from Diploids 1 and 2 were crossed to create MCD1
duplexes (two WT alleles with one G418Rand one HygromicinR
replacement alleles; see Figure 1). Duplexes were transformed with
a URA3 cassette that was targeted to an internal (23 aa in frame)
portion of the open reading frame by amplifying URA3 gene from
pRS306 using primers 59TGGTTACAGAAAATCCTCAACG-
ACC and 59TAAGCATTGATAAACCTTTCAAATAGTGCA-
Ura+transformants were confirmed to be MCD1 simplex if they
maintained G418R, HygromicinR, were non-mating and Tyr+
recombinants could be induced. In addition genomic DNA was
purified from the putative simplex and MCD1 locus was PCR
using the flanking primers 59GTCGAGAAAATCGCGTCTTTC
Since the MCD1 ORF size is almost identical to the G418R
cassette, another set of PCR derived constructs was developed
using a primer within the cassette (59CGTACGCTGCAGGTC-
GAC) and a primer outside the ORF (59AGAAAATTTC-
GGCTTCACCG). This analysis revealed two PCR products
corresponding to G418Rand HygromicinRcassettes. Immediately
after PCR verification of the simplex genotype, patches were
stored at 270uC. At the beginning of each experiment, cells were
streaked from the frozen stock for single colonies which were tested
for the presence of the simplex markers.
SMC3 simplex strain formation
SMC3 simplex strains were created similarly using primers:
ACGCTGCAGGTCGACGGATCCCC and 59CAGTACCTC-
CGAGCTCGTTTTCGA to create G418 and Hygromycin
resistant cassettes directed to the ORF as well as primers
AGCAGATTGTACTGAGAGTGCACC and 59ACCGCGTT-
CGGTATTTCACACCGC to amplify pRS306 to create a URA3
cassette targeted to the ORF. The simplex was verified by primers
flanking the locus: 59 CATCGAAGTGTACACCTGTCACAT
and 59 GAAAAGTAATCTTTTTTGTACGTCG.
RAD50 simplex strain formation
RAD50 simplexes were made in a manner similarly to above.
Oligonucleotides 59TTTCACGGCTTTGCCTTGT and 59TC-
AAAGGTGCTTACGTGCTTG were used to amplify the
flanking region around the RAD50 locus in a null strain from
the Saccharomyces cerevisiae deletion library (G418 cassette replaced
ORF). Both Diploids 1 and 2 were transfected with a G418
cassette and after obtaining heterozygote diploid isolated, the
G418 cassette of one of the diploids was switched to Hygromicin
resistance. The two heterozygote diploids were crossed and the
tetraploid duplex was transformed with a URA3 cassette targeted
to an internal portion of the ORF by amplifying the URA3 gene
from pRS306 using the following primers 59TCTATTCAGGG-
CTGAGAGTGCACC and 59TATCGACCCACTCAATTTGT-
CCGC. Simplex strains were selected as described above.
RAD51 simplex strain formation
Construction of the RAD51 simplex was done by sequential
transfection of Diploid1 with G418 and URA3 cassettes, thereby
replacing two copies of the gene. Diploid 2 was transfected with a
Hygromicin cassette targeted into the ORF of the gene. G418 and
Hygromicin cassettes were made based on strains from a
Saccharomyces cerevisiae deletion library using primers 59 TTGAG-
CATTCCCTGAGCATT and 59TCCCCTAAAAGGATAAAG-
CCG. URA3 cassette was created by amplifying pRS306 using
CACCAGAGCAGATTGTACTGAGAGTGCACC and 59GG-
CTGTGCGGTATTTCACACCGC. The RAD51 simplex strain
was created by crossing the transformed Diploids 1 and 2.
mcd1-1 diploid strain formation
CS1061 and CS1064 (a and a haploids) that contain the same
types of tyr1 truncation alleles described above crossed to yield WT
diploid. Same haploid strains were transformed with an Age1 cut
pVG257  to yield the mcd1-1 strain. The mcd1-1 cells were
verified by sequencing and later crossed to the opposite mating
counterpart to create a diploid mcd1-1 strain.
Western blot analysis
The WT, MCD1 simplex, RAD50 simplex or RAD51 simplex
cells were grown overnight and diluted to fresh media and grown
to for 3 hr in 30uC and then harvested. Before Cells were washed
with double-distilled water and 26107cells were re-suspended in
0.3ml SDS-running buffer, boiled for 10 min and centrifuge
5 minutes 13,000 rpm. Typically 40 ml supernatant was loaded
per lane (corresponding to ,2.56106cells). Following electropho-
resis, the gel was transferred to a membrane using a semi-dry
transfer apparatus for 105 min., according to manufacturer’s
instructions using a PVDF membrane (Invitrogen Carlsbad, CA).
All antibodies were diluted 1:2000 except anti Rad51 that was
diluted 1:5000. Anti Mcd1 antibody was kindly provided by Dr.
Alexander Strunnikov . Anti yRad50 antibody sc32862 and
anti yRad51 antibody sc33626 were from Santa Cruz Biotech-
nology (Santa Cruz, CA). The anti yHistone 3 antibody was
ab1791 from Abcam (Cambridge, MA).
Nocodazole arrest, gamma irradiation, and post
The details of nocodazole arrest, and gamma irradiation have
been described [6,18]. Briefly, nocodazole (20 mg/ml, final
concentration) was added to cells that were growing logarithmi-
cally at 30uC in YPDA media (1% yeast extract, 2% Bacto-
Peptone, 2% dextrose, 60 mg/ml adenine sulfate). G2arrest was
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monitored by cell morphology. Cells were collected by centrifu-
gation, washed and re-suspended in ice-cold sterile water. The cell
suspensions were kept on ice while being irradiated in a137Cs
irradiator (J. L. Shepherd Model 431) at a dose rate of 2.3 krads
per minute. Irradiated cells were harvested by centrifugation and
resuspended in YPDA at 30uC with nocodazole for post-
Pulsed field electrophoresis (PFGE) procedures
PFGE procedures were done as previously described .
Briefly, Contour-clamped Homogeneous Electric Field (CHEF)
systems were used for electrophoresis of yeast chromosomes in this
study. Using a CHEF Mapper XA system (Bio-Rad, Hercules,
CA). These plugs were prepared in 0.5% LE agarose (Seakem,
Rockland, ME) using 1–26107G2-arrested cells per 100 ul plug.
They were cut to a thickness of ,2 mm and loaded in the bottom
of a preparative well so that the entire DNA migrated very close to
the bottom surface of the CHEF gel. PFGE running conditions
were according to the CHEF auto-algorithem separates DNA’s in
the 250–1600 kb range.
Stained-gel, multiple band method for quantitation of
To quantify DSBs in irradiated samples, pulsed-field gels were
stained with SybrGold (Invitrogen, San Diego, CA) and photo-
graphed using a GelLogic200 imaging system (Eastman Kodak,
Rochester, NY). Bands were measured using Kodak MI software
(version 4.0) and the data were exported into Microsoft Excel
(version 11.5.3) for further manipulations to determine DSBs. More
details on the analysis are found in Figure S1A of . Briefly, for
each band corresponding to a complete unbroken Chromosome Y
(any chromosome), the fraction of chromosomes remaining
unbroken (FChrY) after a given dose is simply the net intensity of
the band divided by the net intensity of the corresponding band in
the 0 krad control lane. From the Poisson distribution, the average
number of DSBs (NChrY) is given by the formula:
Plotting the experimentally determined values of N (number of
breaks per chromosome) vs Molecular Weight for each chromo-
some band from a given dose results in an approximate straight
line whose slope is in units of DSBs/mb and is independent of the
total amount of DNA loaded in each lane as long as enough DNA
is loaded for accurate detection of the bands. For details see
reference . The experimentally determined values of the slope
for a given dose are highly reproducible.
PCR identification of cross-over and non cross-over TYR1
Tyr+cells were grown over-night in 30uC in YPDA in deep well
96 plates with shaking. Genomic DNA was purified using DNeasy
of Quiagen (Valencia, CA) and amplified using primers 59
CCA and 59 CACAAAAGAAGGCCTAATATTATAGGAAAT-
comparable growth rates. Overnight cultures of WT and MCD1
simplex cells (2–46107cells/ml) were diluted 100-fold into fresh
MCD1 simplex and WT (tetraploid) strains have
YPDA medium Samples were collected, diluted and plated to
YPDA after 12 and 24 hr. The culture density was calculated at
each time point and the relative increase (compared to time ‘‘0’’)
was determined, results are combined from 4 different cultures of
each genetic background.
Found at: doi:10.1371/journal.pgen.1001006.s001 (0.20 MB TIF)
temperature. The temperature sensitive mcd1-1 diploid cells were
grown at permissive temperature (23uC) and arrested at G2/M
with nocodazole for 3 hr. Survival was determined for cells that
were irradiated, plated to YPDA plates and incubated at semi-
permissive temp (32uC). The plating efficiency without irradiation
of mcd1-1 at 32uC was 35% of that at 23uC. The same procedure
was used with a WT diploid strain where no differences in plating
efficiencies between 32u and 23uC were observed. Results are
combined from 6 cultures of each genetic background.
Found at: doi:10.1371/journal.pgen.1001006.s002 (0.17 MB TIF)
mcd1-1 diploids are sensitive to IR at semi-permissive
is similar for WT diploid and tetraploid cells arrested in G2/M.
Cells were arrested with nocodazole as described in Figure 6
and Materials and Methods and irradiated with the indicated
doses. The data for the tetraploid was taken from Figure 6B (at
least 12 cultures were analyzed). Six diploid cultures were
Found at: doi:10.1371/journal.pgen.1001006.s003 (0.16 MB TIF)
Recombination between homologous chromosome
sensitivity in asynchronous irradiated culture. Six late logarithmi-
cally growing cultures (2–46107cells/ml) of WT and MCD1
simplex cells were diluted 1:20,000 and pronged using a pronging
that delivers1 ml per drop and 121 drops per plates on YPDA
plates (described online at http://m.pu.ru/images/stories/Perfect%
20order%20plating.html). Cells were irradiated at the indicated
doses and colonies were counted after 3 days.
Found at: doi:10.1371/journal.pgen.1001006.s004 (0.22 MB TIF)
MCD1 simplex and WT strains exhibit similar UV
MCD1 simplex strains. Stationary cultures were diluted to fresh
YPDA medium and grown for 3 hr. The culture was divided into
4 equal parts and HU was added to a final concentration of 0, 50,
100 or 150 mM. Cells were grown overnight in the presence of
HU then collected, diluted and spread on to synthetic complete
media. Relative growth inhibition was determined from the
number of colonies arising after the various treatments. This was
the same procedure used to determine TYR1 recombination,
described in Figure 6D.
Found at: doi:10.1371/journal.pgen.1001006.s005 (0.15 MB TIF)
Hydroxurea induced growth inhibition of WT and
induced recombination to sister chromatids in G2 cells, preventing
homologous chromosome events and LOH. Presented are
diagrams for damage-induced recombination in G1and G2cells.
For clarity, events are shown in diploid cells; however, the
concepts extend to tetraploid cells. While gene conversion between
homologous chromosomes in G1cells can lead to homozygosis
over a short region, neither gene conversion nor crossing-over
would lead to extended LOH. In G2cells, gene conversion and/or
crossing-over between sister chromatids does not change the
genetic makeup of cells. However, recombination between
homologous chromosomes can lead to localized changes as found
for G1cells, while crossing-over would lead to LOH, depending on
segregation of the sister chromatids at mitosis. By holding sister
chromatids together, cohesin could direct damage-induced
recombination and repair towards sisters thereby preventing
Proposed role for cohesin in restricting damage-
Reduced Cohesin Enhances Induced Recombination
PLoS Genetics | www.plosgenetics.org 14July 2010 | Volume 6 | Issue 7 | e1001006
Found at: doi:10.1371/journal.pgen.1001006.s006 (0.74 MB TIF)
Generation of TYR1+recombinants by reciprocal
exchange between TYR1 heteroalelles in tetraploid cells. Recip-
rocal exchange (RE) between homologous chromosomes can
occur before (G1) or after (G2) replication of the TYR1 locus. For
the case of G2cells, half the Tyr+(TYR1) cells that underwent
reciprocal exchange would have the ‘‘y’’ allele and half would not,
assuming equal segregation of the sister chromatids. For
recombinants induced in G1, all TYR1+recombinants due to RE
would contain the ‘‘y’’ allele.
Found at: doi:10.1371/journal.pgen.1001006.s007 (0.28 MB TIF)
We thank Alexander Strunnikov and Vincent Guacci for anti-Mcd1
antibodies. We thank Victoria Beja-Glasser for technical support as well as
Jim Mason, Jana E. Stone, and Juan Lucas Argueso for critically reviewing
the manuscript. The many discussions with members of the lab are greatly
Conceived and designed the experiments: SC JWW DAG MAR.
Performed the experiments: SC JWW. Analyzed the data: SC JWW
DAG MAR. Contributed reagents/materials/analysis tools: SC JWW.
Wrote the paper: SC DAG MAR.
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