Increased Genome Instability and Telomere Length in the elg1-Deficient Saccharomyces cerevisiae Mutant Are Regulated by S-Phase Checkpoints

Gross chromosomal rearrangements (GCRs) are frequently observedin cancer cells. Abnormalities in different DNA metabolism includingDNA replication, cell cycle checkpoints, chromatin remodeling,telomere maintenance, and DNA recombination and repair causeGCRs in Saccharomyces cerevisiae. Recently, we used genome-widescreening to identify several genes the deletion of which increasesGCRs in S. cerevisiae. Elg1, which was discovered during thisscreening, functions in DNA replication by participating inan alternative replication factor complex. Here we further characterizethe GCR suppression mechanisms observed in the elg1 ${Delta}$ mutant strainin conjunction with the telomere maintenance role of Elg1. Theelg1 ${Delta}$ mutation enhanced spontaneous DNA damage and resulted inGCR formation. However, DNA damage due to inactivation of Elg1activates the intra-S checkpoints, which suppress further GCRformation. The intra-S checkpoints activated by the elg1 ${Delta}$ mutationalso suppress GCR formation in strains defective in the DNAreplication checkpoint. Lastly, the elg1 ${Delta}$ mutation increasestelomere size independently of other previously known telomeremaintenance proteins such as the telomerase inhibitor Pif1 orthe telomere size regulator Rif1. The increase in telomere lengthcaused by the elg1 ${Delta}$ mutation was suppressed by a defect in theDNA replication checkpoint, which suggests that DNA replicationsurveillance by Dpb11-Mec1/Tel1-Dun1 also has an important rolein telomere length regulation.

INTRODUCTION

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Introduction

Different types of genomic instabilities have been observedin many cancers (16, 20, 44). High levels of chromosome rearrangementsknown as gross chromosomal rearrangements (GCRs), such as translocations,deletion of a chromosome arm, interstitial deletions, inversions,and gene amplification, have been reported in many differentcancers (25, 34). Such a high level of GCRs, which is one ofthe mutator phenotypes, leads to further accumulation of geneticchanges in carcinogenesis (22).

A number of cancer susceptibility syndromes are due to inheritedmutations in genes for DNA damage responses and DNA recombination-repair,and genetic defects resulting in higher frequencies of spontaneousand/or DNA damage-induced chromosome aberrations have been documented(1, 6, 9, 15, 36, 41, 43, 48). Some examples include ATM/ATRin ataxia-telangiectasia; Mre11 in the ataxia-telangiectasia-likedisorder; Nbs1 in the Nijmegen breakage syndrome; hChk2 in theLi-Fraumeni syndrome; and Blm, Wrn, and Rts in the Bloom, Werner,and Rothmund-Thomson syndromes, respectively.

A number of studies have suggested that spontaneous GCRs resultfrom errors during DNA replication that possibly lead to stalledor broken replication forks (21, 24, 28, 30, 35, 38, 46, 50).Besides errors during DNA replication, degradation of telomerescaused by abnormal regulation of telomere maintenance can producealternative sources of DNA damage that can be converted to GCRs(4, 16, 26). Although normal levels of DNA damage activate cellcycle checkpoints and consequently DNA repair pathways (23),high levels of DNA damage or defects in cell cycle checkpointsand/or DNA repair induce GCR formation (16).

In order to understand the mechanisms of GCR suppression, quantitativeassays that can measure different GCR events were developedin Saccharomyces cerevisiae (8, 11, 28). At least six differentpathways have been identified for the suppression of GCRs byusing these assays: (i) at least three different cell cyclecheckpoints during DNA replication, including the DNA replicationcheckpoint and two intra-S checkpoints (11, 21, 28, 30, 42);(ii) a recombination pathway known as break-induced replication(26); (iii) pathways that suppress de novo telomere addition(26); (iv) at least two pathways for proper chromatin assembly(31); (v) pathways that prevent chromosome ends from being joinedto each other and to broken DNAs (7, 26, 33); and (vi) a mismatchrepair pathway that prevents recombination between divergentDNA sequences (27).

Recently, we developed a new genome-wide screening method toidentify more GCR suppressor genes and found 10 new genes (39).One of the new GCR suppressor genes, ELG1, encodes a proteinthat is a component of an alternative replication factor complex(RFC) (2, 3, 12, 14, 39). Elg1 was first identified as a suppressorof Ty1 transposon mobility in the yeast genome (37). Recentextensive genome-wide screens also identified ELG1 as an importantgene for different DNA metabolism (2, 3, 12, 14, 39). In addition,the importance of Elg1 in DNA replication is strongly suggestedby physical interactions between Elg1 and proliferating cellnuclear antigen, an accessory clamp protein for DNA polymerase,or Rad27, a flap endonuclease that removes the RNA primer fromthe lagging strand during DNA replication (14). The elg1 ${Delta}$ mutationhas been shown to cause a cell cycle delay, presumably becauseof a high level of DNA damage during replication and increasedsister chromatid exchange (14). This is similar to the sgs1 ${Delta}$ mutant strain, which has a mutation in the yeast Bloom-Wernergene homolog. Furthermore, Elg1 may have some interaction withcell cycle checkpoints on the basis of observations such asthe abnormal activation of yeast Chk2 kinase Rad53 in the elg1mutant strain upon methyl methanesulfonate (MMS) treatment andthe slow S-phase progression of the elg1 mutant strain (2, 14).Mutation of the ELG1 gene also increases telomere length (14,40). These studies suggest that a defect in the ELG1 gene causesmore spontaneous DNA damage, presumably because of a partialdeficiency in DNA replication and/or defects in other typesof DNA metabolism.

In this report, we further characterize how the elg1 ${Delta}$ mutationincreases GCRs and subsequent suppression pathways that repairspontaneous DNA damage generated by Elg1 deficiency. We demonstratethat the elg1 ${Delta}$ mutation enhances DNA damage, which activatesthe intra-S checkpoint for suppression of further GCR formation.In addition, we show that the telomere length increase causedby the elg1 ${Delta}$ mutation can be suppressed by the DNA replicationcheckpoint deficiency, suggesting a new role for the DNA replicationcheckpoint in telomere maintenance. It also has close linkswith mechanisms of GCR suppression by different cell cycle checkpoints.

MATERIALS AND METHODS

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Materials and Methods

General genetic methods. Media used for propagation of strains were as previously described(28, 39). All of the S. cerevisiae strains used were propagatedat 30°C, except for strains containing the dpb11-1 mutation,which were propagated at 25°C. Yeast transformation, isolationof yeast chromosomal DNAs for use as templates in PCRs, andPCRs were performed as previously described (28, 39).

Strains. The strains used in this study for the general GCR assay wereall isogenic to RDKY3615 (MATa ura3-52 leu2 ${Delta}$ 1 trp1 ${Delta}$ 63 his3 ${Delta}$ 200lys2-Bgl hom3-10 ade2 ${Delta}$ 1 ade8 hxt13::URA3), and those used forthe HO-inducible GCR assay were all isogenic to RDKY4624 (MATaura3::KAN HO::hisG leu2 ${Delta}$ 1 trp1 ${Delta}$ 63 his3 ${Delta}$ 200 lys2-Bgl hom3-10 ade2 ${Delta}$ 1ade8 sit1::HO-URA3). All strains were generated by standardPCR-based gene disruption methods, and correct gene disruptionswere verified by PCR assay as described previously (28, 45).The sequences of the primers used to generate disruption cassettesand to confirm the disruption of indicated genes are availableupon request. The elg1 ${Delta}$ sgs1 ${Delta}$ and elg1 ${Delta}$ mre11 ${Delta}$ mutant strains showeda severe growth defect as previously described (14). The detailedgenotypes of the strains used are listed in Table 1.

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TABLE 1. S. cerevisiae strains used in this study^a

Characterization of GCR rates and breakpoints. All GCR rates were determined independently by fluctuation analysisby the method of the median with at least two independent clonesby two or more times with either 5 or 11 cultures for each clone,and the average value is reported as previously described (17,28). The breakpoint spectra from mutants carrying independentrearrangements were determined and classified as previouslydescribed (28, 39). The significance of differences betweenGCR rates was tested with the Mann-Whitney test by using programsavailable at http://faculty.vassar.edu/lowry/vshome.html.

Induction of GCRs by generation of a single DSB or MMS treatment. GCR assays after the induction of a single double-strand break(DSB) by HO endonuclease or after treatment with MMS were performedas previously described (29, 30). Briefly, for the HO-inducibleGCR assay, S. cerevisiae cells were cultured in synthetic dropoutmedium lacking amino acids required for selection of the plasmidsthat contain a galactose-inducible HO endonuclease gene untila cell density of 1 x 10⁷ to 2 x 10⁷/ml was obtained. Cellswere then washed twice with distilled water and incubated furtherfor 5 h in an equal volume of yeast extract-peptone (YP) mediumcontaining 2% (wt/vol) glycerol and 1% succinic acid. Freshlymade 50% galactose was then added to a final concentration of2% to induce HO endonuclease expression, and cells were incubatedfor 2 h. Cells were washed twice with distilled water and suspendedin 10 volumes of YP medium containing 2% glucose (YPD) and incubatedovernight until the culture reached saturation. The cells werethen plated onto YPD plates and FC plates containing both 5-fluorooroticacid (5-FOA; U.S. Biological) and canavanine (Sigma). The frequencyof cells resistant to both drugs was determined. Three to fiveindependent cultures of each strain were used in each experiment,and each experiment was performed at least twice. The averageincrease in the frequency of GCRs relative to the frequencyof wild-type cells carrying control plasmids is reported. Forthe GCR assay after MMS treatment, log-phase cells were washedtwice with distilled water and suspended in a volume of 0.1%MMS (Sigma) equal to the starting culture volume. After 2 hof incubation, the treated cells were washed with distilledwater two times and suspended in 10 times the original volumeof YPD. After overnight culture at 30°C until saturation,the cells were plated onto YPD plates and FC plates to determinethe frequency of GCRs in the same way as in the HO-inducibleGCR assay. The average increase in the frequency of GCRs relativeto the GCR frequency of untreated cells was reported.

Telomere size determination. Telomere size was determined by a conventional XhoI digestionand Southern hybridization method using a telomeric TG repeatas a probe to detect the Y' class of telomeres. ChromosomalDNA from each strain was digested with XhoI (New England Biolabs)and separated by 0.7% agarose gel electrophoresis. Fragmentedand denatured chromosomal DNAs were then transferred onto nitrocellulosemembrane by a capillary transfer method and UV cross-linkedwith the Strata-linker. The ³²P-labeled telomeric TG repeatobtained from pBC6 by a random priming method was used for hybridizationwith chromosomal DNAs on nitrocellulose as previously described(18). After 2 h of hybridization in ExpressHyb hybridizationsolution (BD Bioscience), the nitrocellulose filter was washedstringently and signals were detected with X-ray film (Kodak).

RESULTS

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Results

Previously, we identified 10 new GCR suppressor genes by a genome-widescreen in S. cerevisiae (39). ELG1, which encodes a proteinparticipating in an alternative RFC with Rfc2-5 during DNA replication,was identified as a GCR suppressor during this and other screens(12, 39). Recent studies have suggested that mutations in ELG1increase spontaneous DNA damage during DNA replication (2, 3,14). In order to understand what causes GCR formation in theelg1 ${Delta}$ strain and what suppression mechanisms inhibit furtherGCR formation in the absence of Elg1, we investigated the GCRformation responses of the elg1 ${Delta}$ strain upon exogenous DNA damageand genetic interactions between the elg1 ${Delta}$ mutation and mutationsin other GCR suppressor genes.

Extra DNA damage in the elg1 ${Delta}$ strain significantly increased GCRs. Recent observations suggest enhanced unrepaired DNA damage inthe elg1 ${Delta}$ mutation, including a high frequency of crossing over,chromosome loss, sister chromatid recombination, and enhancedrecombination at direct DNA repeats (3, 14). However, the elg1 ${Delta}$ mutation increased the GCR rate 49-fold compared to that ofthe wild type (Table 2). It is slightly lower compared to thatof strains carrying other GCR mutator genes that function inDNA replication or checkpoints (29, 30).

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TABLE 2. Effect of elg1 ${Delta}$ on the rate of accumulating GCRs in different cell cycle checkpoint-defective strains^a

If this increase in the GCR rate caused by the elg1 ${Delta}$ mutationwere due to suppression by DNA damage checkpoints, further DNAdamage caused by treatment with DNA-damaging agents could potentiallysaturate DNA damage checkpoints and increase GCR formation synergistically.To test this hypothesis, the elg1 ${Delta}$ mutant strain was treatedwith 0.1% MMS and its induction of an increase in the GCR frequencywas determined (Fig. 1A). The DNA damage caused by MMS treatmentin the elg1 ${Delta}$ mutant strain increased the GCR frequency strikinglymore than in the wild type. A similar increase in the GCR frequencyupon MMS treatment was also observed when a single DSB was introducedin chromosome V as further DNA damage in the middle of two negativeselection markers in the GCR assay (Fig. 1B). MMS treatmentdid not change breakpoint spectra compared to spontaneouslygenerated GCRs. However, a single DSB introduction by a HO endonucleaseincreased the interstitial deletion class of GCR formation (Fig.2). Such breakpoint spectra after MMS treatment or introductionof a single DSB were not different between the wild-type andelg1 ${Delta}$ mutant strains (Fig. 2). When an additional elg1 ${Delta}$ mutationwas incorporated into the pif1-m2 mutant strain, which is defectivein Pif1's telomerase inhibition function, no breakpoint spectrumchange was observed (Fig. 2). However, the GCR rate in the pif1-m2elg1 ${Delta}$ mutant strain is much higher than that of a strain carryingeither single mutation (Table 3) (39).

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FIG. 1. DNA damage caused by MMS treatment or a single DSB caused by HO endonuclease induced the GCR formation in the elg1 ${Delta}$ mutant strain. (A) the wild-type (WT) strain or the elg1 ${Delta}$ mutant strain was treated with 0.1% MMS, and the induction of GCR formation was determined as described in Materials and Methods. (B) The GCR frequency in the presence or absence of HO endonuclease was determined as described in Materials and Methods.

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FIG. 2. Sequences at the junction of GCRs in strains carrying the elg1 ${Delta}$ mutation with or without DNA damage. Sequences physically present on the chromosome(s) are underlined. Telomeric sequences added by de novo telomere addition are in lowercase. ${triangledown}$ , deletion of sequences indicated below. The proportion and percentage of each class of GCR are in parentheses. The nucleotide positions of breakpoints coordinated on the basis of the Stanford Saccharomyces Genome Database system are in brackets. One case showed sequences unidentified in the SGD after the breakpoint from the pif1-m2 elg1 ${Delta}$ mutant strain. Two cases showed an insertion of unknown sequences in the wild type, and one case showed an insertion of chromosome III sequences in the elg1 ${Delta}$ mutant strain from HO-induced GCRs.

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TABLE 3. Effect of elg1 ${Delta}$ on the rate of accumulating GCRs in different GCR^a mutator strains

Such strong induction of GCR formation by treatment with furtherDNA-damaging agents was observed in strains defective in differentS-phase checkpoints presumably because of their lack of sensingof unrepaired DNA damage in the cell that causes GCR formation(29, 30). In the elg1 ${Delta}$ mutant strain, extra DNA damage causedby MMS treatment or a single DSB caused by an HO endonucleasegenerates an excess amount of DNA damage that might eventuallysaturate the DNA damage checkpoint capacity. This could be whythere was a strong induction of an increase in the GCR frequencyupon treatment of the elg1 ${Delta}$ mutant strain with a DNA-damagingagent.

Mutation in the ELG1 gene showed different genetic interactions with strains defective in various cell cycle checkpoints. In order to investigate whether the activation of DNA damagecheckpoints, especially the intra-S checkpoint during S phase,is really needed to suppress GCR formation in the absence ofElg1, GCR rates in strains defective in different cell cyclecheckpoint genes with the elg1 ${Delta}$ mutation were measured. Recently,we reported that there are at least three different S-phasecheckpoints functioning for suppression of GCRs (30). Loss ofthe DNA replication checkpoint, which is presumably activatedbecause of stalled replication forks, increased the GCR ratesignificantly. The other two intra-S checkpoint branches thatare controlled redundantly by the Rad24-Rad17-Ddc1-Mec3 complexand Sgs1 also suppress GCRs (10, 30). The elg1 ${Delta}$ mutation increasedthe GCR rate up to 49-fold compared to that of the wild type(Table 2). The dpb11-1 mutation, which creates a defect in theDNA replication checkpoint, did not produce any significantdifferences in the GCR rate compared to that observed in theelg1 ${Delta}$ mutant strain (Table 2). However, this GCR rate of dpb11-1elg1 ${Delta}$ was lower than that of dpb11-1 itself. When the mutationsof the intra-S checkpoint sensors rad24 ${Delta}$ and sgs1 ${Delta}$ were combinedwith the elg1 ${Delta}$ mutation, the GCR rates were synergistically increased(Table 2). The mutation in the MRE11 gene that knocks down bothintra-S checkpoints also synergistically increased the GCR rateobserved in the elg1 ${Delta}$ mutant strain.

In S. cerevisiae, the Mec1 and Tel1 kinases transduce the S-phasecheckpoint signal to downstream target proteins by phosphorylation(23). Three different kinases, Rad53, Chk1, and Dun1, functionas downstream transducers of Mec1 and Tel1 at S-phase checkpoints.Strains carrying mutations in MEC1 and ELG1 increased the GCRrate synergistically (Table 2). However, the tel1 ${Delta}$ elg1 ${Delta}$ mutantstrain did not show any significant difference in the GCR ratecompared to that of the elg1 ${Delta}$ mutant strain. The mutations ingenes encoding two downstream kinases, RAD53 and CHK1, alsosynergistically increased the GCR rate in the elg1 ${Delta}$ mutant strain.However, the large increase in the strain carrying the dun1 ${Delta}$ mutation, which inactivates the Dpb11-mediated DNA replicationcheckpoint for suppression of GCRs, was reduced by the elg1 ${Delta}$ mutation similarly to that of the dpb11-1 elg1 ${Delta}$ mutant strain.Therefore, both Rad24- and Sgs1-dependent intra-S checkpointsthat transmit signals to Mec1-Rad53/Chk1 are important for thesuppression of further GCR formation in the elg1 ${Delta}$ mutant strain.

The additional elg1 ${Delta}$ mutation in the dpb11-1 and dun1 ${Delta}$ mutantstrains, which are defective in the DNA replication checkpoint,reduced GCR rates compared to those of the dpb11-1 and dun1 ${Delta}$ single-mutant strains (Table 2). This could be due to the activatedintra-S checkpoint caused by the elg1 ${Delta}$ mutation. The rad24 ${Delta}$ mutationthat inactivates the intra-S checkpoint in the dpb11-1 elg1 ${Delta}$ or dun1 ${Delta}$ elg1 ${Delta}$ mutant strain increased GCR rates more than didthe dpb11-1 or dun1 ${Delta}$ mutation (Fig. 3).

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FIG. 3. The reduction of the GCR formation rate by the elg1 ${Delta}$ mutation in strains defective in the DNA replication checkpoint is due to the elevated intra-S checkpoint activities. (A) An additional rad24 ${Delta}$ mutation increased the GCR rate of the dpb11-1 elg1 ${Delta}$ mutant strain. (B) An additional rad24 ${Delta}$ mutation increased the GCR rate of the dun1 ${Delta}$ elg1 ${Delta}$ mutant strain.

GCRs caused by loss of Elg1 synergistically increase with mutations in different GCR mutator genes. Mutations in genes encoding other DNA metabolism proteins increasedGCR rates. Mutations in the RAD27 gene, which encodes a flapendonuclease for RNA primer removal from lagging-strand DNAreplication, increased GCR rates significantly (Table 3) (8).However, the mutation in the ELG1 gene, together with the rad27 ${Delta}$ mutation, increased the GCR rate slightly more than did therad27 ${Delta}$ mutation alone. Similarly, the elg1 ${Delta}$ mutation combinedwith the rfa1-t33 mutation, which produces a deficiency in thesingle-stranded DNA binding activity of the RPA protein, increasedthe GCR rate slightly more than did the rfa1-t33 mutation alone.

The yku70 ${Delta}$ mutation, which produces defects in nonhomologousend joining and telomere maintenance, or the mutation in SRS2,encoding a suppressor of general recombination, decreased theGCR rate observed in the elg1 ${Delta}$ mutant strain (Table 3). Inactivationof the chromatin assembly factor I or the replication-couplingassembly factor complex, which functions in chromatin assemblyduring DNA replication and repair, significantly increases GCRformation (31). When the cac1 ${Delta}$ mutation, which inactivates chromatinassembly factor I function, or the asf1 ${Delta}$ mutation, which causesa defect in the replication-coupling assembly factor complex,was combined with the elg1 ${Delta}$ mutation, the GCR rate was synergisticallyincreased (Table 3).

The ELG1 gene was identified as a GCR suppressor gene duringthe pif1 ${Delta}$ -dependent genome-wide screen (39). PIF1 encodes a telomeraseinhibitor, and mutations in the PIF1 gene increased the GCRrate and telomere length (26, 49). The pif1-m2 mutation in theelg1 ${Delta}$ mutant strain increased the GCR rate synergistically (Table3) (39). The est2 ${Delta}$ mutation, which causes telomerase defects,reduced the GCR rate observed in the elg1 ${Delta}$ mutant strain. However,the rif1 ${Delta}$ mutation, which increases telomere size because ofthe defect in telomere size detection, which is normally performedthrough its interaction with Rap1 and Rif2 (47), did not affectthe GCR rate in the elg1 ${Delta}$ mutant strain (Table 3).

Telomere length increase in the elg1 ${Delta}$ mutant strain and its interaction with other mutations. GCRs are caused by defects in at least two different DNA metabolicpathways. One is from replication defects and the other is fromtelomere maintenance defects (16). Although the GCR increasesin the elg1 ${Delta}$ mutant strain seem to be caused mainly by increasedDNA damage during DNA replication, it is possible that DNA damagethrough telomere maintenance defects such as DSBs generatedby chromosome fusion and breakage might be a source of GCRs.In order to understand whether Elg1 functions in telomere maintenance,the telomere size of the elg1 ${Delta}$ mutant strain was compared withthat of a wild-type control (Fig. 4). The mutation in the ELG1gene increased the telomere size. In order to understand howthe mutation in the ELG1 gene increased telomere size, the telomerelengths of elg1 ${Delta}$ mutant strains with other mutations that havebeen shown to affect telomere size were determined (Fig. 4A).When the PIF1 gene is mutated, telomere size is increased (49).The increased telomere size observed in the elg1 ${Delta}$ mutation wassynergistically increased with an additional pif1-m2 mutation(Fig. 4A). The rif1 ${Delta}$ mutation, which increases telomere sizebecause of the defect in telomere size detection (47), alsocaused further telomere size lengthening with the elg1 ${Delta}$ mutation.When the EST2 gene, which encodes the telomerase catalytic subunit(19), was mutated in the elg1 ${Delta}$ mutant strain, the telomere sizewas increased less but the telomere was slightly bigger thanthat of the est2 ${Delta}$ mutant strain. These observations stronglysuggest that the telomere maintenance function of Elg1 is differentfrom Pif1- or Rif1-dependent pathways on the same telomericsubstrate.

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FIG. 4. Telomere length was determined in different strains carrying the elg1 ${Delta}$ mutation. Chromosomal DNAs from each strain were digested with XhoI and hybridized with the telomeric repeat probe that can detect the Y' class of telomeres. (A) Mutations in telomere maintenance genes had synergistic effects on telomere size when combined with the elg1 ${Delta}$ mutation. (B) S-phase checkpoint sensor gene mutations along with the elg1 ${Delta}$ mutation generated different telomere size changes. (C) The elg1 ${Delta}$ mutation in strains defective in S-phase checkpoint transducer genes also changed telomere length differently. (D) An additional mutation in the RAD24 gene in strains carrying elg1 ${Delta}$ and genes functioning in the DNA replication checkpoint slightly decreased telomere size.

It has been shown that the increased level of single-strandoverhang in the telomere is one source of DNA damage that inducesa cell cycle checkpoint. However, the level of single-strandoverhang at the telomere terminus in the elg1 ${Delta}$ mutant straindid not show any difference from that of the wild type (datanot shown).

When we compared the telomere length variation and the GCR ratesof elg1 ${Delta}$ mutant strains with an additional mutation in telomeremaintenance genes, they were well correlated, except for therif1 ${Delta}$ mutation. For example, the pif1-m2 elg1 ${Delta}$ double mutation,which increased the telomere size synergistically, also increasedthe GCR rate synergistically (Table 2 and Fig. 4A). Similarly,the elg1 ${Delta}$ est2 ${Delta}$ double mutation, which decreased the telomeresize observed in the elg1 ${Delta}$ mutant strain, decreased the GCR rateof that strain. In order to understand whether telomere sizeaffected by the elg1 ${Delta}$ mutation has a close relationship withGCR formation, the telomere length of the elg1 ${Delta}$ mutant straincarrying mutations in checkpoint genes was investigated. Whereasa strain carrying a mutation in an intra-S checkpoint sensorsuch as rad24 ${Delta}$ or sgs1 ${Delta}$ together with the elg1 ${Delta}$ mutation showedan increase in telomere size up to the level of the elg1 ${Delta}$ mutantstrain, the telomere length of the dpb11-1 elg1 ${Delta}$ mutant strainwas not increased (Fig. 4B).

Thus, the telomere length and GCR rate increases induced bythe elg1 ${Delta}$ mutation in strains defective in different checkpointsensors are correlated. Next, the telomere lengths of strainscarrying mutations in the S-phase downstream kinases with theelg1 ${Delta}$ mutation were determined (Fig. 4C). Interestingly, in casesin which the synergistic GCR rate increases were observed (i.e.,rad53 ${Delta}$ and chk1 ${Delta}$ in Table 2), the telomere sizes were increasedby the elg1 ${Delta}$ mutation. The dun1 ${Delta}$ elg1 ${Delta}$ and tel1 ${Delta}$ elg1 ${Delta}$ mutations,which did not increase the GCR rates, also did not produce telomerelengthening. The mec1 mutation, however, is an exception sinceit did not change the telomere length of the strain carryingboth the mec1 ${Delta}$ and elg1 ${Delta}$ mutations despite a synergistic increasein the GCR rate. When an additional mutation in the RAD24 genewas added to the dun1 ${Delta}$ elg1 ${Delta}$ mutant strain, the GCR rate was increasedeven more than that of the dun1 ${Delta}$ strain (Fig. 3). The telomerelength of the dun1 ${Delta}$ elg1 ${Delta}$ rad24 ${Delta}$ mutant strain was not changedor was slightly decreased compared to that of the dun1 ${Delta}$ elg1 ${Delta}$ mutant strain (Fig. 4D). Similarly, telomere size was not changedor was slightly decreased by an additional rad24 ${Delta}$ mutation inthe dpb11-1 elg1 ${Delta}$ mutant strain although the GCR rate was increased(Fig. 3 and 4D).

DISCUSSION

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Discussion

Previously, we identified 10 new GCR mutator genes through genome-widescreening of S. cerevisiae (39). ELG1, one of the GCR mutatorgenes identified, has been documented as a gene functioningin different DNA metabolisms (2, 3, 12, 14, 37, 40). In thepresent study, we further characterized how Elg1 is involvedin the suppression of GCRs and what other mechanisms are activatedin the absence of Elg1 to prevent GCRs. Elg1 functions to suppressspontaneous DNA damage during DNA replication presumably byparticipating in an alternative RFC. The delayed DNA replicationor the increased sister chromatid exchange rates in the elg1 ${Delta}$ mutant strain suggest that there is frequent DNA damage suchas the collapsed DNA replication fork or an increased numberof gaps in the daughter strand (2, 14). It is also possiblethat Elg1 itself functions as a cell cycle checkpoint sensorthe loss of which could increase the chance that DNA damagewill escape proper DNA repair and as a result enhance unrepairedDNA damage to form GCRs.

The strong induction of GCR formation by MMS treatment or asingle DSB caused by an HO endonuclease in the elg1 ${Delta}$ mutant strainstrongly suggests that GCR formation in that strain is due toan increase in DNA damage and further DNA damage saturates theintra-S checkpoints that suppress GCR formation in the elg1 ${Delta}$ mutant strain (Fig. 1 and 5). This conclusion is also supportedby the synergistic GCR rate increase in the elg1 ${Delta}$ mutant straincaused by an additional mutation in chromatin assembly factorgenes that creates more DNA damage (Table 3). The delayed S-phaseprogression observed in the elg1 ${Delta}$ mutant strain (2, 14) supportsthe notion that enhanced DNA damage in the elg1 ${Delta}$ mutant strainactivates the intra-S checkpoint. The synergistic increasesobserved in the strains defective in ELG1 with RAD24, SGS1,MEC1, RAD53, or CHK1 (Table 2) also support the role of an activatedintra-S checkpoint in the suppression of GCRs by repairing DNAdamage in the elg1 ${Delta}$ mutant strain. Therefore, the spontaneousGCR rate of the elg1 ${Delta}$ mutant strain may be lower than those ofother GCR mutators because of the suppression of GCRs by theactivated intra-S checkpoints (Fig. 5). However, since the deficiencyin the intra-S checkpoint also increased the GCR rate in thepresence of Elg1, the mutation in ELG1 could increase spontaneousDNA damage to produce a synergistic GCR rate increase in strainsdefective in the intra-S checkpoint (28, 30).

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FIG. 5. Hypothetical model how Elg1, the intra-S checkpoints, and the DNA replication checkpoint function together to suppress GCRs and telomere elongation. (A) Elg1, the intra-S checkpoint, and the DNA replication checkpoint all function to suppress GCRs at different levels redundantly. Unlike Elg1, which functions to suppress telomere elongation, the DNA replication checkpoint is required for the telomere elongation in the absence of Elg1. The intra-S checkpoint seems also to be involved in telomere elongation (dashed line). (B) In the absence of Elg1, the intra-S checkpoint is highly activated because of DNA damage. This is why the large increase in GCR formation observed in strains defective in the DNA replication checkpoint is suppressed by the elg1 ${Delta}$ mutation. Telomere size is increased because of the lack of Elg1, but in the absence of the DNA replication checkpoint, telomere size was not increased or was only slightly increased by the elg1 ${Delta}$ mutation since the DNA replication checkpoint is required for telomere elongation by the elg1 ${Delta}$ mutation (solid line arrow). An additional mutation in the intra-S checkpoint in strains carrying elg1 ${Delta}$ and a mutation in genes for the DNA replication checkpoint (dpb11-1 and dun1 ${Delta}$ ) further decreased telomere size. This suggests that the intra-S checkpoint also seems to function in telomere elongation in the absence of Elg1 (dashed line arrow).

Inactivation of the DNA replication checkpoint by a mutationsuch as dpb11-1, rfc5-1, or dun1 ${Delta}$ increased the GCR rate significantly(28, 30). However, such increased GCR rates were reduced byan additional elg1 ${Delta}$ mutation in these strains (Table 2 and Fig.3). Thus, the activated intra-S checkpoint due to the elg1 ${Delta}$ mutationmight function to suppress GCR formation in strains defectivein the DNA replication checkpoint (Fig. 5). Supporting thishypothesis is evidence that an additional inactivation of theintra-S checkpoint by the rad24 ${Delta}$ mutation increased the GCR ratesto levels even higher than those observed in the dpb11-1 anddun1 ${Delta}$ mutant strains (Fig. 3). This large increase in GCR ratescaused by an additional rad24 ${Delta}$ mutation in the dpb11-1 elg1 ${Delta}$ ordun1 ${Delta}$ elg1 ${Delta}$ mutant strain is similar to those observed in strainscarrying the rad24 ${Delta}$ dpb11-1 or rad24 ${Delta}$ dun1 ${Delta}$ mutations (28, 30).During DNA replication, a spontaneously stalled replicationfork should be sensed by the DNA replication checkpoint andrepaired (23). However, if there is no DNA replication checkpoint,a stalled replication fork would be collapsed and produce GCRs.In this step, Elg1, which might be enriched at the stalled replicationfork, sometimes opens a channel to GCR formation because ofthe lack of time for recruiting DNA repair machinery. However,if there is no Elg1 along with the DNA replication checkpoint,other RFCs, such as the Rad24-containing RFC that seems to beactivated in the elg1 ${Delta}$ mutant strain, can access DNA damage andproper DNA repair can be performed (Fig. 5).

However, it is also possible that the loss of Elg1 in strainsdefective in the DNA replication checkpoint simply increasesDNA damage, which can cause cells to die even before the generationof GCRs. If this were the case, the dpb11-1 elg1 ${Delta}$ mutant strainwould show growth defects or be more sensitive to DNA-damagingagents than the dpb11-1 mutant strain is. However, the dpb11-1elg1 ${Delta}$ mutant strain did not show any higher sensitivity to manyDNA-damaging agents, including UV, ionizing radiation, MMS,and HU (data not shown). Spontaneous DNA damage such as stalledand/or collapsed replication forks is subject to the DNA replicationcheckpoint to activate DNA repair machinery to suppress GCRs.If the DNA replication checkpoint is not available, cells choosethe intra-S checkpoint for repair. Therefore, in the absenceof Elg1, cells choose the activated intra-S checkpoint to suppressfurther GCR formation.

The telomere size of the elg1 ${Delta}$ mutant strain was larger thanthat of the wild type (Fig. 4), consistent with other results(14, 40). Other known mutations that increased telomere length,including rif1 ${Delta}$ and pif1-m2, synergistically increased telomeresize when combined with the elg1 ${Delta}$ mutation (Fig. 4A). Inactivationof the telomerase catalytic subunit Est2 in the elg1 ${Delta}$ mutantstrain reduced telomere size almost to the est2 ${Delta}$ mutant strainlevel. PIF1 encodes a helicase that functions as an inhibitorof telomerase (26, 49). Rif1 senses the size of the telomereto keep telomere repeats at a certain length through interactionwith the Rap1 and Rif2 proteins (47). The synergistic increasein telomere size in strains carrying the elg1 ${Delta}$ mutation alongwith mutations in these genes suggests that Elg1 functions neitheras a direct regulator of telomerase nor as a telomere size sensor.The increase in telomere length in the elg1 ${Delta}$ mutant strain dependson active telomerase and replication machinery (40; this study),suggesting that Elg1 might participate in replication of lagging-strandsynthesis at the telomere.

Telomere length and GCR rates correlated very well in strainscarrying the elg1 ${Delta}$ mutation. One possible explanation for thiscorrelation is the location of the GCR assay in chromosome V.The GCR assay is located near the telomere and is possibly affectedby telomere size. However, many GCR mutations that actuallydecrease telomere size, such as mre11 ${Delta}$ , rad50 ${Delta}$ , and xrs2 ${Delta}$ , alsoincreased the GCR rate (5, 8, 32). Even other mutations, suchas rif1 ${Delta}$ and rif2 ${Delta}$ , that increased the telomere size did not increasethe GCR rate (26, 47). Therefore, it is very unlikely that acorrelation between telomere size and GCR rates such as thatobserved in strains containing the elg1 ${Delta}$ mutation is due to thelocation of the GCR assay.

The increased telomere length of the elg1 ${Delta}$ mutant strain wasdecreased by DNA replication checkpoint defects (Fig. 4C and Dand 5). Therefore, the DNA replication checkpoint is requiredfor telomere lengthening by the inactivation of Elg1. The slightdecrease in telomere length caused by an additional rad24 ${Delta}$ mutationin the dpb11-1 elg1 ${Delta}$ and dun1 ${Delta}$ elg1 ${Delta}$ mutant strains suggests thatthe activated intra-S checkpoint is at least partially importantto maintain or increase telomere size in strains defective inthe DNA replication checkpoint and Elg1 (Fig. 4D and 5). TheDNA replication checkpoint signaled through Dpb11/Rfc5, Mec1,Tel1, and Dun1 is activated and transduces a signal to downstreamproteins for telomere lengthening or generates time for telomeraseor DNA polymerase further to replicate telomere sequences inthe elg1 ${Delta}$ mutant strain.

Maintaining genome stability is essential for cell growth andsurvival. However, because of mutations in mutator genes orgenotoxic stresses, different types of genome instability aregenerated (16, 20, 44). Such genome instability in mammaliancells further leads to the activation of protooncogenes or inactivationof tumor suppressor genes. As a result, cells are transformedand develop cancer phenotypes. Thus, a clear understanding atthe molecular level of how genome stability is maintained iscrucial for future therapeutic applications against cancer.The present study of the yeast ELG1 gene as a new GCR suppressorgene and its interactions with other GCR suppression mechanismscan shed light on how spontaneous DNA damage that might leadto GCRs is suppressed during DNA replication. We recently clonedthe mammalian homolog of the ELG1 gene, and its role in suppressionof GCRs is under investigation. One intriguing observation isthe deletion of human chromosome 17q11.2 (where human ELG1 islocated) in neurofibromatosis (13), which suggests a putativerole for human ELG1 as a tumor suppressor gene to suppress GCRs.

ACKNOWLEDGMENTS

We thank R. Kolodner (University of California San Diego) forhelpful discussions. We greatly appreciate a scientific editorat the National Human Genome Research Institute, J. Swyers (NationalInstitutes of Health), and members of the K. Myung laboratoryfor comments on the manuscript. K. Myung especially thanks E.Cho.

FOOTNOTES

* Corresponding author. Mailing address: Genome Instability Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Building 49, Room 4A22, Bethesda, MD 20892. Phone: (301) 451-8748. Fax: (301) 402-4929. E-mail: kmyung{at}nhgri.nih.gov.

REFERENCES

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