ploidy dictates repair pathway choice under dna ... · dna repair capacity. little is known about...

Copyright � 2011 by the Genetics Society of AmericaDOI: 10.1534/genetics.110.125450

Ploidy Dictates Repair Pathway Choice under DNA Replication Stress

Xin Chenglin Li*,†,1 and Bik K. Tye*,2

*Department of Molecular Biology and Genetics, College of Agriculture and Life Sciences and †Department of Biomedical Sciences,College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

Manuscript received November 27, 2010Accepted for publication January 11, 2011

ABSTRACT

This study reports an unusual ploidy-specific response to replication stress presented by a defectiveminichromosome maintenance (MCM) helicase allele in yeast. The corresponding mouse allele, Mcm4Chaos3,predisposes mice to mammary gland tumors. While mcm4Chaos3 causes replication stress in both haploid anddiploid yeast, only diploid mutants exhibit G2/M delay, severe genetic instability (GIN), and reducedviability. These different outcomes are associated with distinct repair pathways adopted in haploid anddiploid mutants. Haploid mutants use the Rad6-dependent pathways that resume stalled forks, whereas thediploid mutants use the Rad52- and MRX-dependent pathways that repair double strand breaks. The repairpathway choice is irreversible and not regulated by the availability of repair enzymes. This ploidy effect isindependent of mating type heterozygosity and not further enhanced by increasing ploidy. In summary, adefective MCM helicase causes GIN only in particular cell types. In response to replication stress, early eventsassociated with ploidy dictate the repair pathway choice. This study uncovers a fundamental differencebetween haplophase and diplophase in the maintenance of genome integrity.

AMONG the genetic and epigenetic changes to ge-nomes, changes in ploidy are the most drastic, and

as such, polyploidy is not tolerated by most animal spe-cies (Li et al. 2009a). A recent study of tetraploid yeastsuggests that the deleterious effects of ploidy change aredue to the uncoordinated scaling of the spindle polebody, spindle, and kinetochore, thus resulting in geneticinstability (GIN) (Storchova et al. 2006). However, ploidychanges occur in every sexual cycle of all eukaryotes andare associated with the inclusion or exclusion of an entireset of chromosome homologs that significantly alters theDNA repair capacity. Little is known about whether DNAdamage response is regulated differently in haplophaseand diplophase during sexual cycles.

DNA replication stress, induced by oncogene activa-tion, genotoxic stress, or defects in the DNA replicationmachinery, is believed to cause GIN that acceleratestumorigenesis (Halazonetis et al. 2008). However,DNA replication stress does not always lead to increasedmutation rates or aneuploidy. In metazoans, multiplefactors may affect GIN as a consequence of DNA rep-lication stress but only in some cell types because dif-ferent cells proliferate at different rates under differentcellular contexts (Sarkisian et al. 2007). Therefore, it isdifficult to compare directly the GIN of different cell

types induced by the same replication stress, and dissectthe underlying mechanisms for the differences in GIN.Saccharomyces cerevisiae is an excellent model for studyingthe mechanisms and pathways leading to GIN, and anoften-used model for cell type-specific regulation. Yeastnaturally exists in three cell types: haploids with twomating types, MATa, MATa and MATa/a diploid, whichis the default state in the wild. These cell types have dif-ferent properties, most of which can be attributed to thedifferent genotypes at the mating type locus (Friis andRoman 1968; Durand et al. 1993; Galitski et al. 1999;Barbour and Xiao 2006; Valencia-Burton et al. 2006;Meyer and Bailis 2008).

Repair pathways may be distinctly regulated in differ-ent cell types. Double strand breaks (DSBs) are repairedby two main pathways, nonhomologous end-joining(NHEJ) and homologous recombination (HR), whichhave distinguishable mutagenic potential (Takata et al.1998; P’ques and Haber 1999). Yeast mainly uses theHR pathway. In diploid yeast, NHEJ is severely disabledthrough the repression of NEJ1, a key component ofNHEJ, by the transcriptional repressor, Mata1–Mata2(Frank-Vaillant and Marcand 2001) encoded by theMATa and MATa genes. While human somatic cells useNHEJ as the main pathway to repair DSBs (Mao et al.2008), mouse embryonic stem (ES) cells display en-hanced HR capacity (Shrivastav et al. 2008). Further-more, the choice between NHEJ and HR for DSB repairis also cell cycle regulated through CtIP/Ctp1/Sae2(Limbo et al. 2007; Yun and Hiom 2009). However, littleis known about the cell type-specific regulation of da-mage repair other than DSBs such as those induced by

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.110.125450/DC1.

1Present address: Department of Biochemistry and Molecular Pharma-cology, University of Massachusetts Medical School, Worcester, MA 01605.

2Corresponding author: Department of Molecular Biology and Genetics,College of Agriculture and Life Sciences, Cornell University, Ithaca, NY14853. E-mail: [email protected]

Genetics 187: 1031–1040 (April 2011)

http://www.genetics.org/cgi/content/full/genetics.110.125450/DC1





replication defects (Barbour and Xiao 2006; Shrivastav

et al. 2008; Jain et al. 2009).Mcm4 is a subunit of the hexameric MCM replication

helicase (Bochman and Schwacha 2008). Mcm4Chaos3 isa cancer susceptible allele of Mcm4 that predisposesmice to mammary gland tumors (Shima et al. 2007b).Previously we showed that the effects of Mcm4Chaos3 inmice was recapitulated in diploid yeast strains bearingthe corresponding mutation, allowing us to study themechanism and consequence of replication stress-inducedGIN in yeast (Li et al. 2009b). Our initial study showedthat haploid yeast bearing the mcm4Chaos3 allele was gros-sly normal (Shima et al. 2007a). The unexpected resultwas that GIN and the checkpoint-dependent cell cycledelay was a diploid-specific outcome. Clearly, impor-tant diploid-specific phenotypes may have been over-looked in the past because haploid mutants arecommonly used in yeast genetic studies.

In this study, we show that the mcm4Chaos3 haploid notonly exhibits no cell cycle delay but also no obvious GIN,although both haploid and diploid mutants showevidence of compromised replication. We demonstratethat the different outcomes of replication stress areassociated with distinct repair pathways activated in thehaploid and diploid mutants. The haploid mutants usethe Rad6-dependent pathways that resume stalled forkswhereas the diploid mutants use the Rad52- and MRX-dependent pathways that repair double strand breaks.The repair pathway choice is regulated neither by theavailability of different repair enzymes nor by MAT locusheterozygosity, but rather by ploidy itself. Distinct fromthe effect of geometric constraints shown in polyploids(Storchova et al. 2006), the diploid-specific defectshown here is not enhanced by increased ploidy. Thisstudy reveals a fundamental difference between haplo-phase and diplophase on the maintenance of genomeintegrity. It provides a model to study the cell type-specific GIN outcomes and the repair pathway choicesin response to a cancer susceptible helicase defect.

MATERIALS AND METHODS

Yeast strains and plasmids: Isogenic haploid W303 yeaststrains mcm4Chaos3 were constructed as described (Shima et al.2007a). All strains used in this study are in the W303background and listed in supporting information, Table S1.The strain background carries the rad5-535 point mutation.Replacing this allele with RAD5 had little effect on ploidydifference (data not shown).

The MATa/D and MATD/a diploids were constructed bydisruptions of the MAT locus using the pFP19 plasmid, a giftfrom Hannah Klein. Ectopic expression of NEJ1 was per-formed using PMV01 plasmid with the empty vector PMV04 ascontrol; both plasmids were gifts from James Haber. TheMATa/a haploid (nonmater) was constructed by transformingeither MATa haploid with BE96 or MATa haploid with BE97.The BE96 and BE97 plasmids were gifts from Hannah Klein.

Flow cytometric analysis: Approximately 1 3 107 cells werecollected from log-phase cultures and processed as described

(Clarke et al. 2001). DNA was stained with Sytox Green(Molecular Probes, Eugene, OR) and profiles were analyzedusing a Becton Dickinson (San Jose, CA) LSR II with a 530/30BPchannel filter and BD FACS DiVa software Becton Dickinson.

Growth curve and doubling time: Saturated cell cultureswere diluted 25 times in YPD medium and then grown at 30�for 24 hr. The absorbance at 600 nm was measured every10 min using the microplate reader Tecan M200. Growth ratesand doubling times were calculated by the maximum slopeplotted in log scale. For each experiment where doublingtimes of different strains were compared, all strains wereprocessed simultaneously in three independent trials thatroutinely showed variations in doubling times of ,0.1 hr.

Intrachromosomal recombination assay: Each strain carriedthe recombination reporter leu2-riTURA3Tleu2-bsteii, whichhad a heteroallelic duplication of LEU2, with URA3 insertedbetween the two LEU2 genes. Gene conversion was deter-mined by fluctuation tests measuring Leu1 Ura1 frequency.The deletion frequency was determined by fluctuation testsmeasuring fluoro-orotic acid resistance rates. Each test wasperformed with 10 colonies and repeated twice for each strain(Xu et al. 2004).

Determination of spontaneous mutation frequency: Theforward mutation rate at the CAN1 1ocus was determined bystandard methods (Sia et al. 1997), using at least 12 indepen-dent cultures for each rate estimate. Frequencies were calcu-lated from the occurrence of canavanine-resistant mutantsusing the method of the median (Lea and Coulson 1949).

Measurement of gross chromosome rearrangements: Thetest strains carry a marker�10 kb from the telomere of ChrXV-Lto select for gross chromosome rearrangements (GCR) eventsthat result from break-induced replication in repairing DSBs.The GCR rate was measured on the basis of the previouslyreported protocol (Kanellis et al. 2007). Ten colonies fromeach strain were tested, and two rounds of independent ex-periments were conducted.

Cell viability: Cell viabilities were measured by first countinglog phase cells in a hemacytometer before plating in triplicateon YEPD and counting visible colonies after 3 days of growth atpermissive temperatures.

Mitotic recombination assay: A standard assay for measur-ing mitotic recombination and chromosome loss was used(Hartwell and Smith 1985). The test strain was heterozy-gous for mutations in CAN1 and HOM3, two markers locatedon opposite arms of chromosome V. The haploid strain withthe can1 mutation was resistant to canavanine (Canr) and thehom3 strain was auxotrophic for threonine (Thr�). Heterozy-gous diploid strains were Cans and Thr1. Mitotic recombina-tion was scored by the Canr Thr1 phenotype. Over 90% of theCanr strains scored were Thr1.

Pulsed-field gel electrophoresis: Standard pulsed-field gelelectrophoresis (PFGE) procedures were used according tothe manufacturer’s instructions (Bio-Rad). Agarose plugscontaining 3 3 108 cells/ml were loaded onto a 1% agarosegel in 0.5 3 Tris–borate EDTA (TBE) buffer and electro-phoresed at 6 V at an angle of 120� for 22 hr at 14� with aninitial switch time of 50 sec and a final switch time of 90 sec.

RESULTS

Unusual ploidy effect: Haploid mcm4Chaos3 mutantsare grossly normal without G2/M delay or obviousGIN: We showed previously that mcm4Chaos3/Chaos3 homo-zygotes and mcm4Chaos3/D hemizygotes display a G2/Mdelay prior to anaphase. The G2/M delay depended onthe DNA damage checkpoint gene RAD9 (Li et al.

1032 X. C. Li and B. K. Tye

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2009b). However, in the mcm4Chaos3 haploid no cell cycledelay was observed (Figure 1A). The haploid mutant wasindistinguishable from wild type with respect to dou-bling time (2.66 6 0.02 vs. 2.62 6 0.03 hr).

We previously showed that the diploid mutant dis-played severe GIN with a 100-fold increase in mitoticrecombination. We investigated the recombination ratein the mcm4Chaos3 haploid strain using a recombinationreporter that measured intrachromosomal gene con-version and deletions between direct repeats (Xu et al.2004). We found that the haploid mcm4Chaos3 mutant hadwild-type levels of deletion events (6.18 6 1.96 3 10�5 vs.8.57 6 1.95 3 10�5) and gene conversion (1.93 6 0.48 3

10�5 vs. 1.11 6 0.19 3 10�5). Thus, the haploid mcm4Chaos3

mutant did not display hyperrecombination.We next examined the mutation frequency of mcm4Chaos3

haploid using the CAN1 forward mutation assay (Kokoska

et al. 2000). Haploid mcm4Chaos3 only showed a subtlemutator phenotype, with a mutation frequency (1.1 6

0.2 3 10�6) about 2.5-fold above wild type (3.9 6 0.1 3

10�7). The slight increase of the mutation frequency in themcm4Chaos3 haploid prompted us to examine the potentialincrease of GCR frequency in the ChrXV-L GCR strain(Kanellis et al. 2007). In this assay the loss of two sele-

ctable markers, the CAN1 and URA3 genes, �10 kb fromthe telomere of ChrXV-L was measured. We observed nodramatic increase of GCR in the mutant strain (10.0 6

0.9 3 10�8) in comparison to wild type (6.0 6 1.0 3 10�8).In summary, mcm4Chaos3 causes a cell cycle delay and

severe GIN in diploid but not in haploid yeast. It ispossible that the haploid mutant has a lower tolerancefor GIN, and haploid cells experiencing mutations re-adily die. To test this possibility, we compared theviabilities of the haploid and diploid mutants. We foundthat the haploid mutant (86 6 5%) had a better viabilitythan the diploid mutant (59 6 2.5%) (Li et al. 2009b).We also tested the possibility that chromosome aberra-tions or other mutations may suppress the growth defectin mcm4Chaos3 haploids. We examined the karyotype bygenomic DNA comparative hybridization microarray(CGH), and did not find any aberrations (data notshown). We backcrossed the mcm4Chaos3 haploid to wild-type background and did not find the segregation of anygrowth defects (data not shown). Furthermore, defectsof homozygous diploid mutants constructed by the ma-ting of haploids were rescued by introducing a wild-typeMCM4 (Li et al. 2009b). These disparate observationsbetween the haploid and homozygous diploid mutant

Figure 1.—The normal growth of mcm4Chaos3 haploid requires intact checkpoint functions and fork stabilization proteins. (A)The mutant (mcm4C) and wild-type (H-WT) haploid were arrested with a factor and released to fresh medium. Cell cycle progres-sion was monitored by FACS at 20-min intervals. (B and C) mcm4Chaos3 shows synthetic growth defects with rad9D (B) and mec1D (C).Tenfold serial dilutions of cells from each strain were spotted on complete medium plates and incubated at 37�. The fold changesin growth rate of mutant vs. wild type and double mutant vs. single mutant are shown on the right. (D, E, F, and G) mcm4Chaos3 showssynthetic lethality with fork destabilizing mutations mrc1D and tof1D at 37� in haplophase (D and E) and diplophase (F and G). Thefold change in growth rate between mutant and wild type is shown on the right. H-WT, haploid wild type; D-WT, diploid wild type.

Cell Type-Specific Genetic Instability 1033

suggest an unusual dependence on ploidy for themanifestation of GIN stimulated by the mcm4Chaos3 allele.

mcm4Chaos3 haploid requires intact checkpointfunctions for normal growth: To investigate whetherdamage is also induced by mcm4Chaos3 in haploids, weconstructed double mutants of mcm4Chaos3 with variouscheckpoint mutations. Since the defects of mcm4Chaos3

diploids are more severe at 37� (Li et al. 2009b), we usedthe higher temperature to impose greater stress on thehaploid double mutants. If mcm4Chaos3 caused insufficientdamage to activate checkpoint response, then the check-point response pathways would be dispensable. Con-versely, if mcm4Chaos3 caused significant DNA damage inhaploids, then cells with checkpoint mutations would failto detect the damage generated by mcm4Chaos3, resultingin unrepaired damage and severe growth defects. Sup-porting the second possibility, mcm4Chaos3 showed syntheticgrowth defects with rad9D and mec1D, respectively (Fig-ure 1, B and C), and the cell cycle distributions were alsoaltered in the double mutants (Fig. S1A). These resultsindicate that mcm4Chaos3 also induces DNA damage inhaploids.

Both haploid and diploid mutants suffer fromreplication stress: MCM helicase has been shown tomigrate with the elongation fork during DNA replica-tion (Aparicio et al. 1997). To investigate whethermcm4Chaos3 mutant has compromised replication forks,we constructed double mutants of mcm4Chaos3 with mrc1D

and tof1D, respectively. Mrc1 and Tof1 are replicationfork stabilization proteins that are loaded onto DNAshortly after replication initiation and travel with theelongating fork (Katou et al. 2003). The synergisticgrowth defects of mcm4Chaos3 with mrc1D and tof1D (Figure1, D–G) in both haploid and diploid suggest thatmcm4Chaos3 causes replication fork defects that requirefork stabilization in both cell types. However, thissynthetic effect seems to be more severe in the haplo-phase (Figure 1, D and E) than in the diplophase(Figure 1, F and G).

Our results suggest that both haploid and diploidmutants suffer from replication stress and require thecooperation of fork stabilization proteins for survival. Ifboth haploid and diploid mutants experience similarstress, then the ploidy difference may be caused bydistinct downstream repair mechanisms that are morerobust and efficient in haploid cells such that forkdamages are repaired without causing GIN.

The diploid mutant requires the HR-dependent DSBrepair pathway, while the haploid mutant does not:Considering the 100-fold increase in mitotic recombi-nation observed in the mcm4Chaos3/Chaos3 diploid strain (Li

et al. 2009b), recombination-mediated replication is alikely mechanism for repairing the replication defects inthe diploid mutant. To visualize the state of the ch-romosomes in the absence of damage repair, we placedmcm4Chaos3 into a recombination-deficient background(mcm4Chaos3/Chaso3 rad51D/D). The double mutant showed

synthetic lethality at 37� (Figure 2A), arresting withabout 4C DNA (Figure 2B). Fragmentation of chromo-somes is observed on contour-clamped homogeneouselectric fields (CHEF) gel (Figure S1B). This resultindicates that homologous recombination (HR) is indis-pensable in the diploid for repairing DNA damageinduced by mcm4Chaos3.

Three principal lesions are able to trigger spontane-ous HR: DSBs, stalled replication forks, and collapsedforks (Saleh-Gohari et al. 2005). DSB recognition andkinase activation of ATM/Tel1p are mediated through theMre11-Rad50-Xrs2 (MRX) protein complex (Costanzo

et al. 2004). An increase in DSBs would result in a greaterrequirement for MRX function. Consistent with this idea,we found that mcm4Chaos3 and rad50D were syntheticallylethal at 37� in the diplophase (Figure 2C). A similar effectwas also observed in mcm4Chaos3 with mre11D (Figure S2A).Therefore, double strand break repair (DSBR) is requiredfor the mcm4Chaos3 diploid. The requirement for DSBR, thefragmentation of chromosomes (Figure S1B), the RAD9 -dependent cell cycle delay, and the increased aneuploidy(Li et al. 2009b) strongly suggest that DSBs are formed inthe diploid mutant.

Another DSB repair pathway independent of HR isNHEJ, which is sequestered in diploids (Valencia et al.2001). It is likely that DSBs are also generated in themcm4Chaos3 haploid, but NHEJ is the more efficient path-way for preventing DSBs from translating into GIN. Totest this hypothesis, we constructed double and triplemutants of mcm4Chaos3 with DSB repair mutations inhaploid yeast. Dnl4 (DNA ligase IV) is a key componentof NHEJ pathway (Martin et al. 1999). The doublemutant of mcm4Chaos3 with dnl4D or rad50D grew as well aswild type in the haplophase (Figure 2, D and E). Dis-ruption of both the HR and the NHEJ pathways did notshow synergistic defects with mcm4Chaos3 in the triple mu-tant (Figure 2E), suggesting that the haploid mutantdoes not require DSBR under the stress of the mcm4Chaos3

mutation. The dispensability of DSBR and the absenceof a cell cycle delay (Figure 1A) suggest that the haploidmutant does not experience DSBs as the diploid mutantdoes, but experiences damage other than DSBs.

The haploid mutant requires the RAD6-dependentstalled fork resumption pathway, while the diploidmutant does not: We suspected that stalled forks are theprimary spontaneous damage in the mcm4Chaos3 mutants.Other than HR, cells may also resume replication atstalled forks via the RAD6 -dependent pathway and thenovel MGS1-dependent pathway (Barbour and Xiao

2003). To dissect these repair pathways in mcm4Chaos3

haploids, we constructed double and triple mutants ofmcm4Chaos3 with mutations defective in repairing stalledforks. There was no observable synthetic growth defectin mcm4Chaos3 mgs1D rad51D (Fig. S2B), though mcm4Chaos3

showed synthetic growth defect with rad6D (Figure 2F)(Fan et al. 1996; Ulrich and Jentsch 2000). In contrast,the diploid mutant did not require the RAD6 -dependent





pathway (Figure 2G). Therefore, unlike the diploid mu-tant that relies solely on the DSBR pathway for survival,the haploid mutant is able to resume replication of thestalled forks presumably before the stalled forks de-generate into DSBs.

Damage in the diploid mutant cannot be repaired bythe NHEJ pathway: The diploid mutant showed a forkdefect that required Mrc1 and Tof1 for fork stability(Figure 1, F and G) while the haploid showed spontane-ous damage that manifested as stalled forks. It is likely thatthe substrates that activate DSBR in the diploid mutantmay not be the conventional DSBs but may have beenderived from collapsed forks. A fork collapse produces aone-ended DSB that has no second end with which torejoin, and therefore not a substrate for NHEJ pathway(Shrivastav et al. 2008). To test this hypothesis, weactivated the NHEJ pathways in the diploid by ectopicallyexpressing the NEJ1 gene, which hypothetically wouldrescue the lethality of the mcm4Chaos3 rad52D doublemutant if two-ended DSBs were created (Valencia et al.2001). However, the activation of NHEJ did not repair thedamage of the mcm4Chaos3 diploid (Figure 3A). Also, we didnot observe on CHEF gel DNA smears that are indicativeof spontaneous DSBs in the mcm4Chaos3 diploid mutant(Figure S2C), suggesting that the damage in mcm4Chaos3

diploid mutant is not conventional DSBs but collapsedstalled forks. The repair of collapsed forks in diploidwould have a less stringent requirement for fork stabiliz-ing proteins compared to stalled forks in haploid (Figure1, D–G). Thus, stalled forks and collapsed forks (or one-ended DSBs) may be two outcomes of the replicationstress caused by mcm4Chaos3 and they activate differentdownstream repair pathways (Figure 3E).

In summary, haploid and diploid yeast use distinctrepair pathways to restore the replication defectscreated by mcm4Chaos3, resulting in dichotomous out-comes of GIN. What determines the choice of differentrepair pathways? Is it determined by the availability ofrepair pathway on an ad hoc basis or by an upstream-regulated process? To investigate whether haploid anddiploid mutants could be coerced into using alternativerepair pathways to repair their fork defects, we alteredthe availability of repair pathways in the haplophase anddiplophase in the next set of experiments.

The choice of repair pathway is not reversible ordetermined by the availability of repair proteins: Thefork resumption pathway used in the mcm4Chaos3 haploidis also available in diploid (Barbour and Xiao 2006).The fact that mcm4Chaos3 is synthetically lethal with rad52D

(Figure 3A) indicates that the damage generated in the

Figure 2.—Distinct repair pathways are used in haploid and diploid mutants. (A) The mcm4Chaos3/Chaos3 rad51D/D double mutantshows synthetic conditional lethality at 37�. (B) FACS profile shows that most cells are arrested with�4C DNA when mcm4Chaos3/Chaos3

rad51D/D mutant was grown to log phase at 30� and then shifted to 37� for 3 hr. (C) The diploid mcm4Chaos3/Chaos3 rad50D/D doublemutant shows synthetic lethality at 37�. (D) The haploid mcm4Chaos3 rad50D double mutant shows normal growth. (E) The haploidmcm4Chaos3 dnl4D double mutant shows normal growth. The mcm4Chaos3 does not show additional synthetic effect with dnl4D orrad52D. (F) The haploid mcm4Chaos3 rad6D double mutant show synthetic growth defect. (G) The diploid mcm4Chaos3 rad6D doublemutant does not show synthetic growth defect.



diplophase could not be channeled to other pathwaysfor repair when the HR pathway was blocked. Thus, thechoice of using the HR pathway was not regulated by theavailability of repair pathways.

Two DNA helicases, Sgs1 and Srs2, regulate the repairpathways that resume stalled forks (Barbour and Xiao

2003). Sgs1 and Srs2 process recombination intermedi-

ates formed during fork stalling and channel theaberrant structures into the RAD6 - dependent path-way for repair. Indeed, as previously reported, sgs1D

and srs2D appear to cause rampant recombination inhaploids (Figure 3, B and C) (Gangloff et al. 2000). Toinvestigate whether HR pathway is able to repair theDNA damage in the mcm4Chaos3 haploid, we introducedsgs1D and srs2D, respectively, into the haploid mutantstrain to channel damage repair from the fork re-sumption pathway to HR. The mcm4Chaos3 srs2D haploidmutant showed synthetic growth defects at the restric-tive temperature (Figure 3B). The effect of the mcm4Chaos3

and sgs1D double mutant was even more dramatic,showing synthetic lethality at the restrictive temperature(Figure 3C, see similar effect in mcm4Chaos3 top3D, FigureS2D) and arresting at late S or G2/M phase (Figure 3D)with fragmented chromosomes (Figure S1B). This syn-thetic lethality is suppressed by the deletion of RAD51(Figure 3C), suggesting that repairing the DNA damagesubstrate created in sgs1D mcm4Chaos3 haploid by HR maybe the cause of lethality. Thus, although the HR pathwayis available, this pathway is unable to repair the DNAdamage in haploid.

In summary, haploid and diploid mutants use distinctand noninterchangeable repair pathways to repair theirfork defects (Figure 3E). (The dissection of additionalrepair pathways is shown in Figure S3.) This distinct andexclusive choice of repair pathways is most likelyregulated by some upstream mechanism that is associ-ated with haploid and diploid identities. The two ob-vious determinants for cell-type identity are mating typeheterozygosity and ploidy itself.

The diploid-specific effect is not due to MAT locusheterozygosity: Diploid yeast strains are more resistantthan haploid strains to g-rays, UV, and methyl meth-anesulphonate (MMS). This resistance is partly due toheterozygosity at the MAT locus (Heude and Fabre

1993; Barbour and Xiao 2006). The effect of MATheterozygosity on increased resistance to DNA damageagents is dependent on the function of HR proteins(Saeki et al. 1980). To investigate whether the unusualdiploid specificity of the mcm4Chaos3 phenotype is due toheterozygosity at the MAT locus, we constructed thedouble mutant mcm4Chaos3 rad51D in a haploid with MATheterozygosity. The mcm4Chaos3 rad51D diploid was lethalat the restrictive temperature (Figure 2A) while thehaploid double mutant was grossly normal (Figure 4A).Notably, the MATa/a mcm4Chaos3 rad51D haploid did notshow any synthetic effect (Figure 4A). Therefore, thediploid-specific defect is not due to MAT heterozygosity.

To investigate further the role of MAT heterozygosityin this diploid-specific defect, we constructed MATa/D

and MATD/a diploids. The mcm4Chaos3 diploid that washemizygous at the MAT locus showed chromosomefragmentation (Figure S1B) and was inviable at therestrictive temperature (Figure 4B), indicating that thedamage could no longer be repaired. The observation

Figure 3.—The haploid and diploid mutants use differentrepair pathways to counter damages induced by mcm4Chaos3.(A) The diploid mcm4Chaos3/Chaos3 rad52D/D double mutant showssynthetic lethality at 37�, and this lethality cannot be rescued byectopic expression of NEJ1 with empty vector as control. (B)mcm4Chaos3 shows synthetic growth defects with srs2 mutant.(C) mcm4Chaos3 shows synthetic lethality with sgs1D at 37�, andthis lethality is rescued by destroying the HR pathway withrad51D. (D) sgs1D mcm4Chaos3 double mutant is grown to logphase at 30� and then shifted to 37� for 3 hr. FACS profile showsthat most cells were arrested with 2C DNA. (E) Schematic sum-mary illustrating the choice of repair pathway is not intercon-vertible, but dictated by the repair substrate processed in aploidy-dependent manner. Mating type heterozygosity regu-lates the HR pathway used in diploid as shown in Figure 4.







that the growth defect was worse in diploids withhemizygous MAT than with heterozygous MAT suggeststhat the diploid-specific growth defects are not due toMAT heterozygosity. Rather, MAT heterozygosity is re-quired for viability in mcm4Chaos3 diploid presumablybecause of its role in upregulating the HR pathway, aspreviously reported (Valencia-Burton et al. 2006).

To investigate the effect of MAT heterozygosity inrepair pathway choice in the mcm4Chaos3 diploid, we mea-sured the loss of heterozygosity (LOH) frequency ofCAN1 with respect to HOM3 on the left arm ofchromosome V (Hartwell and Smith 1985) at thepermissive temperature. There was little difference inthe LOH frequency between MATa/a mcm4Chaos3/Chaos3

(2.60 6 1.60 3 10�3) and MATD/a mcm4Chaos3/Chaos3

(1.02 6 0.49 3 10�3) strains, which was �100-foldelevated over that of the wild type (2.12 6 0.11 3

10�5) (Li et al. 2009b). Nearly all LOH events in MATD/a

mcm4Chaos3/Chaos3 were also due to mitotic recombination asthey were in MATa/a mcm4Chaos3/Chaos3 (Li et al. 2009b). Evenwhen the ability to perform HR was compromised in theMATD/a background (Figure 4B) (Valencia-Burton

et al. 2006), the damage was still committed to HR repairindependent of MAT heterozygosity, consistent with theprevious observation (Figure 3E) that the repair pathwaychoice is not regulated by the availability of repairenzymes.

In sum, the diploid-specific defects and the repairpathway choice are not determined by MAT heterozygos-ity or the availability of repair enzymes, but by the ploidy.

The diploid-specific defects do not increaseproportionally with ploidy: The diploid-specific defectcould be the effect of an increase in spontaneous DNAdamage associated with the replication of an extra

chromosome set or the geometric constraints in scalingthe mitotic spindle with increased ploidy (Storchova

et al. 2006). To investigate these possibilities, we con-structed the triploid and tetraploid mutant and wild-type strains by mating MATa/D diploid with MATa

haploid or MATD/a diploids. We found that none ofthe diploid-specific defects such as G2/M delay (Figure5A), growth defect (Figure 5B), or increased doublingtime (Figure 5C) are significantly enhanced with in-creased ploidy. We also constructed triploids andtetraploid mutants containing only one copy of MATaor MATa (with disrupted MAT heterozygosity). Similarto the diploid mutant, heterozygosity of the MAT locuswas required for viability (Figure 5D), suggesting thatthe triploid and tetraploid cells may also use HR torepair the mcm4Chaos3-induced DNA damage. This resultsuggests that the ploidy effect in mcm4Chaos3 mutant eitheris not due to geometric scaling (Storchova et al. 2006)or has reached the threshold in the diploid.

DISCUSSION

Choice of repair pathway is under active controlrather than passive shunting: Cells develop multiplepathways to repair particular types of DNA damage.These pathways are distinct with regard to repairefficiency and mutagenic potential and must be tightlycontrolled to preserve viability and genomic stability. Inthis study, although the fork resumption pathways andthe DSB repair pathways are available in both haploidand diploid, the choice is dictated by ploidy. We haveshown that the haploid and diploid mutants are unableto use each other’s designated repair pathways to repairtheir replication defects (Figure 3) and that these repairpathways do not randomly compete for substrates on anad hoc basis.

There is substantial evidence that the choice of repairpathway may be passively shunted in particular cell typeson the basis of availability (Gudmundsdottir et al.2007). Why did our study reach a different conclusionfrom previous studies? We believe that the disparity liesin the difference in the initial processing of thespontaneous damage generated in mcm4Chaos3 haploidand diploid. These processed structures define thesubstrates for the repair pathway and dictate the repairpathway choice. In the diploid mutant, the spontaneousdamage seems to be processed to DSBs, while the samedamage is rescued directly without degenerating intoDSBs in the haploid (Figure 3E). Our results reveal acomplex upstream regulation of repair pathways underreplication stress, which may be at least as complex asthe end processing of DSBs (Mimitou and Symington

2008).Cell type-specific GIN: Cell type-specific GIN is often

a shunned topic because of its complexity. Themcm4Chaos3 yeast provides a tangible model to dissectthe underlying causes. The haploid and diploid yeast

Figure 4.—The diploid-specific growth defect is not causedby mating type heterozygosity. (A) The MATa/a mcm4Chaos3

rad51D haploid mutant was grossly normal compared toMATa/a rad51D, a mcm4Chaos3 rad51D and a rad51D haploidmutant. (B) MATa/D and MATD/a mcm4Chaos3 diploids werelethal at 37�, while growth of the MATa/D and MATD/awild-type diploids appear normal.


are congenic (except for the mating type locus), can begrown in the same conditions, and suffer similarly fromendogenous replication defects. These inherent prop-erties allow us to investigate the difference in DNAdamage response specific to cell-type identity between

haploids and diploids. In diploid yeast, we previouslyshowed that mcm4Chaos3 resulted in LOH and that a hy-permutable subpopulation gained new traits such asaneuploidy and improved growth (Li et al. 2009b).Here, we showed that the haploid mcm4Chaos3 mutanthad no growth defect or obvious GIN, although it alsosuffered from replication stress. This difference inoutcome is associated with the regulation of repairpathways in different cell types. In the diploid mutant,the error prone HR repair pathway seemed to be thecause of hyperrecombination and aneuploidy, while inthe haploid, the RAD6 - dependent pathway repaired forkdamage with sufficient fidelity without causing GIN.

HR is generally less mutagenic than NHEJ (Takata

et al. 1998; P’ques and Haber 1999). However, inrepairing mcm4Chaos3-induced DNA damage, HR in dip-loid is much more error prone than the fork resumptionpathway in haploid. This difference in GIN may be duenot to the repair pathway used but to the manner inwhich the initial damage is processed. It is known thatDSBs pose the greatest challenge for cells in maintain-ing genome integrity. While replication defects in thediploid mutant are processed to DSBs, the haploidmutant managed to avoid the formation of DSBs. WhenDSBR is operating at a high level (100-fold abovenormal), even the HR pathway will generate severe GIN.

Haploid and diploid yeast have fundamentaldifferences: The dichotomous response to replicationstress indicates that there are fundamental differencesbetween haploid and diploid yeast. In the mcm4Chaos3

diploid, MAT heterozygosity confers the availability ofthe HR repair pathway (Figure 4B), but does not ob-ligate the commitment of this repair pathway. Interest-ingly, under replication stress ploidy determines therepair pathway choice and the consequent GIN. Thisploidy effect may have evolved as another layer of re-gulation to ensure genome integrity during changes inploidy associated with the sexual cycle. The diploid-specific GIN and cell cycle delay seem to be unique tothe mcm4Chaos3 allele, but have not been observed in othergenotoxic stresses (data not shown) (Heude and Fabre

1993; Barbour and Xiao 2006). This apparentlyunique response could either be due to the under-investigation of replication stress in diploids or thechallenges of a specific helicase defect. In particular,defective replicative helicases are not known to exposesingle strand DNA and may have to activate the intra-Sphase checkpoint differently (Branzei and Foiani

2005).If the ploidy effect observed in this study is not due to

geometric scaling, it may act through the dosagechanges of certain genes. In yeast, ploidy-regulatedgene expression has been observed for the G1 cyclins,Flo11 (Galitski et al. 1999), components of the cell wall(De Godoy et al. 2008), and cell surface proteins (Wu

et al. 2010). However, these genomic scale comparisonsbetween haploids and diploids were conducted in

Figure 5.—The diploid-specific defects are not propor-tional to ploidy. (A) FACS profile of the triploid and tetra-ploid mutant. (B) Growth defects on YPD plate of triploidand tetraploid wild type and mcm4Chaos3 mutant. (C) Compar-ison of the doubling time (in hours) of wild type and mutantin different ploidy background expressed as fold increase.(D) MATD/D/a mcm4Chaos3 triploids are lethal at 37�, whilethe MATD/D/a wild-type triploids also grow poorly comparedto triploids with mating type heterozygosity. This plate wasovergrown for 4 days to see the difference between MATD/D/a mcm4Chaos3 triploid and MATD/D/a wild-type triploid.


normal conditions (Galitski et al. 1999; De Godoy et al.2008; Wu et al. 2010) and therefore not designed toidentify the genes responsible for ploidy-specific stressresponse. The mechanisms that regulate homologousrecombination for DSBR are conserved from yeast tohuman (Hartwell and Smith 1985; P’ques andHaber 1999; Limbo et al. 2007; Yun and Hiom 2009).We believe that the mechanisms that regulate repairpathway choices in response to replication stress mayalso be evolutionarily conserved.

We thank Eric Alani and John Schimenti for discussions. Thiswork is supported by National Institutes of Health GM-072557 awardedto B.K.T.

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Communicating editor: F. Winston


GENETICSSupporting Information


Ploidy Dictates Repair Pathway Choice under DNA Replication Stress

Xin Chenglin Li and Bik K. Tye

Copyright � 2011 by the Genetics Society of AmericaDOI: 10.1534/genetics.110.125450

X. C. Li and B. K. Tye 2 SI

A

B

rad9 mcm4C

rad9 mec1 mec1 mcm4C

sgs1mcm4

C

MATa/mcm4

C/Cmcm4C/C

MAT /mcm4

C/C

rad51 / sgs1mcm4

C

MATa/mcm4

C/Cmcm4

C/C

MAT /

mcm4C/C

rad51 /

30°C 37°C

FIGURE S1.—Evidence of DNA damage caused by mcm4Chaos3 allele. A) The cell cycle distribution of cells in rad9 , mcm4Chaos3

rad9 , mec1 and mcm4Chaos3 mec1 strains. Double mutants of mcm4Chaos3 combined with various checkpoint mutations have

significantly different cell cycle distributions. rad9 : G1/early S (42%), late S/G2 (46%); mcm4Chaos3 rad9 : G1/early S (32.5%),

late S/G2 (54.5%); mec1 : G1/early S (22.2%), late S/G2 (61%); mcm4Chaos3 mec1 : G1/early S (32.6%), late S/G2 (57%). B)


CHEF gel analysis of log-phase culture of mcm4Chaos3/Chaos3 rad51 / diploid mutant, mcm4Chaos3 sgs1 haploid mutant, MATa/

mcm4Chaos3/Chaos3 and MAT / mcm4Chaos3/Chaos3 diploid mutant at 30°C, and after shift to 37°C for three hours. DNA

trappedinthewell is incompletelyreplicated chromosomes of S phase cells. Significant chromosome smears were observed in

mcm4Chaos3 mutant in repair defective background at 37°C.


D-WT

mre11 /

mre11 /

mcm4C/C

mcm4C/C

30°C 37°C

30°C 37°C

H-WT

mre11

mre11

mcm4C

mcm4C

H-WT

mgs1

mcm4C

rad51

mgs1 rad51

30°C 37°C30°C 37°C

A

C

D-WT mcm4C/

B

H-WT

top3

top3

mcm4C

mcm4C

30°C 37°CD

FIGURE S2.—Characterization of repair pathway and the lesion in mcm4Chaos3 mutant. A) The diploid mcm4Chaos3/Chaos3 mre11 /

double mutant shows synthetic lethality at 37°C (left), while the haploid mcm4Chaos3 mre11 double mutant shows normal growth

(right). B) There is no synthetic effect of mcm4Chaos3 with mgs1 rad51 in haploid. C) CHEF gel analysis of diploid wildtype and

diploid mcm4Chaos3/ log phase culture at 30°C. The mcm4Chaos3/ has a stronger defect than mcm4Chaos3/Chaos3 (Li et al., 2009a). There

is no obvious smears as indicatives of DSBs. D) The haploid mcm4Chaos3 top3 double mutant shows synthetic lethality at 37°C.


H-WT

rev1

rev1

mcm4C

mcm4C

H-WT

rev3

rev3

mcm4C

mcm4C

H-WT

rad30

rad30

mcm4C

mcm4C

30°C 37°C

30°C 37°C

30°C 37°C

D-WT

rev3 /

rev3 /

mcm4C/C

mcm4C/C

30°C 37°C

30°C 37°CD-WT

rad30 /

rad30 /

mcm4C/C

mcm4C/C

D-WT

rev1 /

rev1 /

mcm4C/C

mcm4C/C

30°C 37°C

B

FIGURE S3.—Dissection of the requirement of other members of the RAD52 epistasis group and the requirement for

translesion synthesis pathway in the mcm4Chaos3 mutant. A) There is no synthetic effect of mcm4Chaos3 with rad54 in haploid, but

there is a modest effect of mcm4Chaos3 with rad54 in diploid, indicating an important but not indispensible function of Rad54 in HR

repair pathway in the diploid mutant. There is no synthetic effect of mcm4Chaos3 with rad55 or rad57 in haploid or diploid. B)

There is no synthetic effect of mcm4Chaos3 with rev1 , rev3 , or rad30 in haploid or diploid.


TABLE S1

Strain list

Strain Genotype Source

W303 (haploid) MATa ade2-1 can1-100 his 3-11,15 leu2-3, 112 trp1-1 ura 3-1 rad5

W303 (diploid)

MATa/MAT ade2/ade2 his3/his3 leu2/leu2 trp1/trp1 ura3/ura3

can1/can1 rad5/rad5

XLY047 MATa mcm4Chaos3

This lab

XLY495 mcm4Chaos3/Chaos3

This lab

XLY891 MATa leu2RI::URA3::leu2BstEII

XLY892 MATa leu2RI::URA3::leu2BstEII mcm4Chaos3

Derived from strain 1538-2C from

Hannah Klein Lab

XLY142 MATa CAN1

XLY141 MATa CAN1 mcm4Chaos3

Derived from MC42-2d from Tom

Pete lab

XLY619 MAT ChrXV 10KB::CAN1-URA3

XLY620

XLY621

MAT ChrXV 10KB::CAN1-URA3 mcm4Chaos3

Derived from strain yDD1775 from

Daniel Durocher Lab

XLY10 MATa sml1::URA3 mrc1::HIS3

XLY37 MATa sml1::URA3 mrc1::HIS3 mcm4Chaos3

XLY38 MAT sml1::URA3 mrc1::HIS3 mcm4Chaos3

XLY90

XLY91

mrc1::HIS3/mrc1:HIS3 sml1::URA3/sml1::URA3

XLY92

XLY93

mrc1::HIS3/mrc1:HIS3 sml1::URA3/sml1::URA3 mcm4Chaos3/Chaos3

Derived from strain YJT134 from

John Diffley Lab

XLY54 MATa rad50::hisG-URA3-hisG

XLY192 MAT rad50::hisG-URA3-hisG

XLY193 MATa rad50::hisG-URA3-hisG mcm4Chaos3

XLY194 MAT rad50::hisG-URA3-hisG mcm4Chaos3

Derived from strain KKY604-17C

from Hannah Klein Lab

XLY94

XLY95

rad50::null/rad50::null Cross between XLY54 and XLY192

XLY96

XLY97

rad50::null/rad50::null mcm4Chaos3/Chaos3

Cross between XLY193 and

XLY194

XLY236 rad52::TRP1/rad52::TRP1

XLY237 rad52::TRP1/rad52::TRP1 mcm4Chaos3/Chaos3

Derived from strain KKY614-10B


XLY232 MATa sgs1::URA3

XLY233 MATa sgs1::URA3 mcm4Chaos3

Derived from strain KKY1958-10A


XLY641 MATa sgs1::URA3 rad51::HIS3

XLY643 MATa sgs1::URA3 rad51::HIS3 mcm4Chaos3

XLY336 MATa tof1::URA3

XLY337 MAT tof1::URA3

XLY338 MATa tof1::URA3 mcm4Chaos3

XLY339 MAT tof1::URA3 mcm4Chaos3

Derived from strain YHG307 from

Rolf Sternglanz Lab


XLY425

XLY426

tof1::URA3/tof1::URA3 Cross between XLY336 and

XLY337

XLY427

XLY428

tof1::URA3/tof1::URA3 mcm4Chaos3/Chaos3


XLY339

XLY168 MAT rad6::LEU2

XLY170 MATa rad6::LEU2 mcm4Chaos3

XLY171 MATa rad6::LEU2

XLY172 MATa rad6::LEU2 mcm4Chaos3

Derived from strain M31 from

Takashi Hishida Lab

XLY661

XLY662

rad6::LEU2/ rad6::LEU2 Cross between XLY168 and

XLY171

XLY663

XLY664

rad6::LEU2/ rad6::LEU2 mcm4Chaos3/Chaos3


XLY172

XLY49 MAT rad51::HIS3

XLY113 MAT rad51::HIS3 mcm4Chaos3

XLYY82 rad51::HIS3/rad51::HIS3

XLYY83 rad51::HIS3/rad51::HIS3 mcm4Chaos3/Chaos3

Derived from strain KHKY1039-4D


XLY157 MATa srs2::HIS3

XLY158 MAT srs2::HIS3

XLY159 MATa srs2::HIS3 mcm4Chaos3

XLY160 MAT srs2::HIS3 mcm4Chaos3

Derived from strain KKY590-1D


XLY299 MAT dnl4::URA3

XLY300 MATa dnl4::URA3

XLY301 MATa dnl4::URA3 mcm4Chaos3

XLY302 MAT dnl4::URA3 mcm4Chaos3

Derived from strain 1186-5C from

Hannah Klein Lab

XLY303 MATa dnl4::URA3 rad52::TRP1

XLY304 MATa dnl4::URA3 rad52::TRP1 mcm4Chaos3

XLY305 MAT dnl4::URA3 rad52::TRP1 mcm4Chaos3

XLY18 MATa sml1::URA3 mec1::LEU2

XLY19 MATa sml1::URA3 mec1::LEU2 mcm4Chaos3

Derived from strain YJT74 from

John Diffley Lab

XLY161 MAT rad9::URA3

XLY163 MAT rad9::URA3 mcm4Chaos3

Derived from strain 3834 from Judith

Berman's lab

XLY274 MAT / diploid

XLY276 MAT / diploid mcm4Chaos3/Chaos3

XLY277 MATa/ diploid

XLY280 MATa/ diploid mcm4Chaos3/Chaos3

XLY827 hom3-10/HOM3 can1-100/CAN1 mcm4Chaos3/Chaos3

XLY6 MAT / diploid hom3-10/HOM3 can1-100/CAN1 mcm4Chaos3/Chaos3

Derived from MC42-2d and

HLK1042-1C from Tom Petes lab

XLY347

XLY348

Triploid wildtype

XLY349

XLY350

mcm4Chaos3/Chaos3/Chaos3


XLY351 Tetraploid wildtype

XLY353

XLY354

mcm4Chaos3/Chaos3/Chaos3/Chaos3

XLY126 MATa mgs1::LEU2 rad51::HIS3

XLY137 MATa mgs1::LEU2 rad51::HIS3 mcm4Chaos3

XLY136 MAT mgs1::LEU2 rad51::HIS3 mcm4Chaos3

Derived from strain TH201 from

Takashi Hishida Lab

XLY146 MATa mre11::LEU2

XLY147 MAT mre11::LEU2

XLY144 MATa mre11::LEU2 mcm4Chaos3

XLY148 MAT mre11::LEU2 mcm4Chaos3

Derived from strain LSY569 from

Hannah Klein Lab

XLY120

XLY121

mre11::LEU2/mre11::LEU2 Cross between XLY022 and

XLY023

XLY122

XLY123

mre11::LEU2/mre11::LEU2 mcm4Chaos3/Chaos3


XLY025

XLY369

XLY370

MAT / / Triploid wildtype

XLY371

XLY372

MAT / / mcm4Chaos3/Chaos3/Chaos3

XLY178 MATa top3::LEU2

XLY179 MATa top3::LEU2 mcm4Chaos3

Derived from strain KKY606-1A


XLY258 MATa rad54:: HIS3 mcm4Chaos3

Derived from strain HKY624 from

Hannah Klein Lab

XLY261 MAT rad54:: LEU2 mcm4Chaos3

Derived from strain HKY596-2B


XLY254

XLY255

rad54:: HIS3/ rad54:: LEU2 Cross between HKY624 and

HKY596-2B

XLY256

XLY257

rad54:: HIS3/ rad54:: LEU2

mcm4Chaos3/Chaos3


XLY261

XLY285 MATa rad55:: LEU2

XLY284 MAT rad55:: LEU2

XLY286 MATa rad55:: LEU2 mcm4Chaos3


Derived from strain HKY597-2C


XLY357

XLY358

rad55:: LEU2/ rad55:: LEU2 Cross between XLY284 and

XLY285

XLY359

XLY360

rad55:: LEU2/ rad55:: LEU2

mcm4Chaos3/Chaos3


XLY287

XLY288 MATa rad57:: LEU2

XLY289 MAT rad57:: LEU2

XLY290 MATa rad57:: LEU2 mcm4Chaos3


Derived from strain HKY598-8B


XLY365 rad57:: LEU2/ rad57:: LEU2 Cross between XLY288 and


XLY366 XLY289

XLY367

XLY368

rad57:: LEU2/ rad57:: LEU2

mcm4Chaos3/Chaos3


XLY291

XLY622 MATa rad30:: HIS3

XLY602 MAT rad30:: HIS3

XLY623 MATa rad30:: HIS3 mcm4Chaos3

XLY624 MAT rad30:: HIS3 mcm4Chaos3

Derived from strain T145 from Roger

Woodgate Lab

XLY611

XLY612

rad30::HIS3/ rad30::HIS3 Cross between XLY622 and

XLY602

XLY613

XLY614

rad30::HIS3/ rad30::HIS3

mcm4Chaos3/Chaos3


XLY624

XLY603 MATa rev1:: HIS3

XLY605 MAT rev1:: HIS3

XLY606 MATa rev1:: HIS3 mcm4Chaos3

XLY604 MAT rev1:: HIS3 mcm4Chaos3


Woodgate Lab

XLY628

XLY629

rev1::HIS3/ rev1::HIS3 Cross between XLY603 and

XLY605

XLY630

XLY631

rev1::HIS3/ rev1::HIS3

mcm4Chaos3/Chaos3


XLY604

XLY608 MATa rev3:: URA3

XLY601 MAT rev3:: URA3

XLY610 MATa rev3:: URA3 mcm4Chaos3

XLY609 MAT rev3:: URA3 mcm4Chaos3


Woodgate Lab

XLY615

XLY616

rev3:: URA3/ rev3:: URA3 Cross between XLY608 and

XLY601

XLY617

XLY618

rev3:: URA3/ rev3:: URA3

mcm4Chaos3/Chaos3


XLY610

XLY507 mcm4Chaos3/

ploidy dictates repair pathway choice under dna ... · dna repair capacity. little is known about...

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