INTRODUCTION

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In the yeast Saccharomyces cerevisiae, chromosomal double-strand breaks (DSBs) are predominantly repaired by a recombinational repair pathway involving interaction between the broken molecule and a homologous chromosome or sister chromatid. This repair pathway is largely dependent on the RAD52 epistasis group gene products (56, 61). To a lesser extent and in the absence of a homologous template, yeast can repair plasmid or chromosomal DSBs using a nonhomologous, end-joining (NHEJ) repair pathway that depends on YKU70, YKU80, and the MRE11-RAD50-XRS2 complex as well as DNL4 and LIF1 (summarized in references 40, 41, 76, 79, and 80).

The products of the SIR genes have also been proposed to function in DSB repair and NHEJ, but their role is unclear. The Sir2, Sir3, and Sir4 proteins form a complex that has a direct role in producing transcriptionally repressed chromatin structures at both telomeres and the silent mating type loci HML and HMR. Recent studies using immunolocalization and immunoprecipitation have suggested that DSB damage leads to the dissociation of Sir and Ku proteins from telomeres and relocation in complexes at the DSB site (48, 49, 50). Furthermore, the enhanced sensitivity to -ray or methyl methanesulfonate (MMS) damage in the absence of Sir4 supports a direct role for this protein in the toleration of DSBs (48, 80, 81).

Although some studies using plasmid end-joining assays have reported a direct effect of the Sir4 gene product on NHEJ repair (29, 79, 80), other studies have shown little or no direct effect of the Sir gene products on NHEJ (5, 39). The latter studies suggest that, in some strains, NHEJ repair is inhibited to similar extents in sir a1/2 haploids and diploids, in which the transcriptional regulators a1 and 2 are expressed within both MATa and MAT (5, 73). In sir cells, expression of these regulators from within the silent mating loci HML and HMR determines the diploid mating type and confers an a1/2 haploid phenotype. Furthermore, enhanced resistance to the killing effects of ionizing radiation is observed in MATa/MAT heterozygous diploids compared to the killing effects in either MATa/MATa or MAT/MAT homozygous diploids (24, 43). These results indicate that a gene(s) under MAT regulation influences both NHEJ and recombinational repair pathways in yeast.

Both haploid Rad⁺ cells in the G₁ phase of the cell cycle as well as diploid rad52 cells are sensitive to ionizing radiation, and it appears that approximately one unrepaired DSB is sufficient to kill these cells (62). It has been assumed that death results from the direct effects of an unrepaired DSB, namely, the loss of essential genetic material. However, in a previous study (25), one or a small number of unrepaired DSBs induced by ionizing radiation caused killing in polyploid rad52 strains, suggesting that lethality need not be due simply to the direct effects of an unrepaired DSB.

Unrepaired lesions, particularly DSBs, can have a severe impact on cells, and this has led to studies examining their effects on cell cycle progression and viability. The underlying mechanisms appear to be relevant to many disease processes (20, 83). DNA damage, including unrepaired DSBs, can lead to a transient arrest of cells in the G₁, S (2, 70), and G₂ (22, 84, 85, 86) phases of the cell cycle. This arrest involves many genes that act in multiple pathways in the budding yeast Saccharomyces cerevisiae (2, 26, 69, 86). The RAD9, RAD17, RAD24, MEC3, and SFP1 genes are required for arrest in the G₂ phase of the cell cycle (22, 26, 44, 85, 86, 89). Specific kinases, including MEC1 (rad3⁺ of Schizosaccharomyces pombe) and MEC2 (SAD1 or RAD53), are required for arrest in S and G₂ phases (30, 75, 86). RAD9 and RAD24 genes are also required for arrest in the G₁ phase of the cell cycle (70). The RAD9, RAD17, RAD24, and DUN2 genes are also required for arrest in the S phase of the cell cycle following DNA damage (54, 58). These checkpoint control genes appear to function in a complex network to assess the integrity of chromosomal DNA and delay cell progression after damage, thereby increasing the opportunity for DNA repair.

While a DSB produced in asynchronously growing cells leads to G₂ arrest, cells can eventually proceed past the G₂ checkpoint and undergo mitosis even if the damage is not repaired. This toleration mechanism has been termed adaptation (20, 38, 58, 78, 83) and has been observed in cells that experienced a single unrepairable DSB in a dispensable plasmid, chromosome, or artificial chromosome derived from lambda DNA or human DNA (60, 65, 67). Both CDC5 and CKB2 have been shown to be important for adaptation in a rad52 mutant (78). A single enzymatically induced DSB in a dispensable disomic chromosome that remained unrepaired, due to the absence of the RAD52 recombinational repair pathway, caused permanent G₂ arrest in strains defective in either of these genes (78). These results must be interpreted with regard to other effects that RAD52 mutations might have on DNA metabolism (6), including cell cycle arrest and subsequent growth inhibition. Recently, it was shown that the long G₂ arrest induced in yku70 mutants by a DSB in an essential chromosome can be suppressed by rad50 or mre11 deletions or by a mutation in the single-strand binding protein RPA (38).

We have taken an alternative approach to identifying genes that influence cell cycle progression in response to unrepaired DSBs. Previously, we had shown that, in Rad⁺ cells, the presence of a single DSB, which is not subject to recombinational repair, could lead to cell death (7, 8, 9). Induction of a single unrepairable DSB by HO endonuclease at a YZ target site in a dispensable plasmid or yeast artificial chromosome (YAC) caused prolonged but transient G₂ arrest. Subsequently, many of the cells slowly adapted to the persistent damage, resumed cycling, and formed microcolonies (<50 cells) which did not progress further. Since the DNA containing the DSB was dispensable (7, 8, 9), this process was called indirect lethality because the cells died by a mechanism(s) that does not involve direct loss of essential chromosomal material. While a persistent DSB in dispensable DNA always results in G₂ arrest, in some strain backgrounds such as LS20 (60, 65, 67) there was no reduced viability from the unrepaired DSB. This finding indicates that there may be genetic differences in the types of adaptation to DSB damage. The CDC5 and CKB2 adaptation genes were identified in a repair-deficient rad52 version of the LS20 strain (78).

We initiated a screen utilizing our DSB break system in a repair-proficient LS20 strain to identify adaptation mutants that exhibit extended G₂ delay and indirect lethality. We found that the SIR genes play an important and indirect role in the adaptation response to persistent DSB damage. Sir⁺ cells adapted quickly to a persistent DSB and rapidly formed viable microcolonies after G₂ arrest. The single unrepaired DSB led to a more prolonged G₂ arrest in cells that are deficient for SIR2, SIR3, or SIR4. In contrast to the adaptation mutants described previously (cdc5 and ckb2 mutants), these sir Rad⁺ mutant cells eventually adapted to the damage and slowly divided. They produced mainly microcolonies that did not proceed further, resulting in clonal death. Toleration of the persistent YAC DSB does not appear to be due to a direct effect of SIR gene products on the NHEJ repair functions at the DSB site since the responses were not affected by RAD50. Checkpoint adaptation and viability were restored in sir2, sir3, and sir4 strains with the silent mating type loci HML and HMR deleted, but viability was not reduced in Sir⁺ cells that express the a1 and 2 transcriptional regulators, which confer an a1/2 haploid phenotype. Therefore, there are at least two functions of the SIR genes that determine the biological effects of a persistent, HO-induced DSB. One role is indirect in that SIR is required for silencing HMR and HML, which can regulate a gene(s) that is involved in checkpoint adaptation and survival following induction of a persistent DSB. However, this effect can occur only in the absence of SIR4, suggesting that SIR can have an additional role, possible through direct interactions at the break site not involving end joining.

	MATERIALS AND METHODS

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Strains, plasmids, and YACs. The haploid S. cerevisiae strain LS20 (Table 1) (65) was transformed (66) with the selectable (URA3) low-copy-number plasmid (YZ-CEN) that contains a 45-bp HO endonuclease target sequence (YZ) flanked by nonhomologous DNA sequences (7). In strain LS20 the HO endonuclease was fused to the GAL promoter and integrated at the ade3 locus (65, 67). Strain LS20 was also spheroplasted and transformed with the u8 and u17 YACs, which contain human DNA (8, 9), by methods previously described (36). The u8 YAC is a 365-kb derivative of YAC12 with the YZ site (and URA3 marker) positioned 70 kb from the telomere within the human DNA. The u17 YAC is a derivative of YAC12 with a 230-kb terminal deletion and with the YZ site (and URA3 marker) positioned 5 kb from the telomere within the human DNA. These YACs are hereinafter collectively termed YZ YACs. Strain LS20 containing the VS8 lambda DNA-based YAC has also been described previously (67). In strains CBY and NR85 (Table 1) the GAL::HO fusion was on the centromere plasmid pGALHOT, as previously described (7, 8, 9). Strain NR85 was transformed with the VS8 YAC by methods previously described (36). Strains CBY and NR85 are both Sir⁺. A series of isogenic strains were constructed using strain LS20 containing the u8 YAC (see below). These strains and their relevant genotypes have been listed in Table 1.

MAT

Survival. To determine the effects of a HO-induced DSB, cells were grown to log phase in synthetic complete (SC)-glucose (GLU) medium (88) with selection for the individual YACs or plasmids. Selection for the pGALHOT TRP1 plasmid in strains CBY and NR85 was maintained throughout the experiment. (The differences in a strain's ability to express indirect lethality [CBY and NR85 versus LS20] was not due to selection for the pGALHOT plasmid in CBY and NR85, since LS20 containing pGALHOT and YZ-CEN exhibited high levels of survival on galactose (GAL) when selection for pGALHOT was maintained [data not shown].) Growth in GLU medium repressed HO expression, and the YZ vectors remained intact. Cells were then washed in sugarless SC medium and plated to SC-GLU and SC-GAL plates. Survival was determined by a strain's colony-forming ability on GAL compared to that on GLU plates after 72 h of incubation at 30°C. No selection was placed on the YZ YACs (8, 9) or the YZ-CEN (7) plasmid so that they would be dispensable following HO induction of a DSB at the YZ target site. HO-induced cutting at the YZ site was an efficient process since most of the surviving colonies (>95%) did not retain the YAC or plasmid URA3 marker, as determined by replica plating (data not shown).

Commitment to death and kinetics of marker loss following induction of a DSB. To determine the times at which the URA3 marker was lost and cells were committed to death, cells were grown and washed as described above. In the pullback experiments, cells were resuspended in SC medium-uracil (URA)-sugar and aliquots of between 200 and 300 µl were delivered to plastic petri dishes. Cells were imbedded in agar by adding 15 ml of liquid SC medium plus 2% GAL with or without URA agar medium (45°C). After various periods of incubation at 30°C, we added a 15-ml overlay containing 2% agar and SC plus 4% GLU with or without URA. The diffusion of GLU into the bottom layer rapidly repressed the expression of HO endonuclease.

Library screening. The LS20 strain containing the selectable (URA3) YZ-CEN plasmid was transformed with a high-copy-number, selectable (LEU2) yeast genomic library (ATCC 37323 [53]), and 3,100 transformants were examined. Since this library contained genomic fragments ranging from 5 to 20 kb in length, this screen corresponded to coverage of the yeast genome one to two times. Library transformants were grown overnight in 96-chambered multiwell dishes in 200 µl of SC-GLU medium lacking URA and leucine. Approximately 2 µl from each well was placed with a 48-pin pronged device on either SC-GLU or SC-GAL plates lacking leucine to maintain selection for the library plasmids. Candidate factors were identified by their ability to cause slow growth (and possibly lethality) on GAL following induction of a DSB in the YZ-CEN plasmid.

Precise deletion of the YZ sites within HML and HMR. The YZ sites within the HMR and HML loci of LS20 strains were deleted using a two-step procedure. HMR was first deleted using a PCR-mediated disruption procedure. The primers HMR-Z (5'-GAAAGATAAA CAACCTCCGC CACGACCACA CTCTATAAGG CCAAATGTAC AAACACATCT TCCCAAATATC CAGAGCAGAT TGTACTGAGA GTGCACC-3') and HMR-Y (5'-GAGTTTGGGT ATGTAATATG AGAATCAAAC TTAAATATAT CCTATACTAA CAATTTGTAG TTCATAAATA CGCATCTGTG CGGTATTTCA CACCGC-3') were obtained from Bioserve Biotechnologies and contain sequences that closely flank the YZ site within the Y and Z domains of HMR as well as sequences (italics) homologous to plasmid pRS314 (71) that flank the TRP1 marker contained within that plasmid. Following PCR amplification of TRP1 within pRS314, the LS20 or LS20 sir4 strains containing u8 were transformed with the PCR amplification product. Deletion of the YZ junction within HMR was confirmed by Southern blotting (data not shown). The YZ junction within HML was then disrupted. The primers HML-Z3G (5'-CTTCCCAATA TCCGTCACCA CGTACTTCAG CATAATTATT CGTCAACCAC TCTACAAAAC CAAAACCAGGG CGTACGCTGCCAGGTCGAC-3') and HML-Y3G (5'-CACAGTTTGG CTCCGGTGTA AAACAAAATG TCTTGTCTTC TCTGCTCGCT GAAGAATGGC ACGCGGACAA ATCGATGAAT TCGAGCTCG-3') were also obtained from Bioserve Biotechnologies and contain sequences flanking the YZ junction within the Y and Z domains of HML as well as sequences (italics) that flank the G418 marker within plasmid pFA6a (82). Following PCR amplification of the G418-resistant (G418^r) marker in plasmid pFA6a, strains containing the u8 YAC and the deleted YZ site within HMR were transformed with the PCR product. Transformants were selected on plates containing G418 as described previously (82), and disruption of HML was confirmed by Southern blot analysis (data not shown).

One-step deletion of HML and HMR through targeted PCR-mediated circularization of chromosome III. The LS20 strain containing the u8 YAC was completely deleted for the HML and HMR loci using a one-step, PCR-mediated targeted deletion that resulted in circularization of chromosome III. To do this, primers derived from sequences obtained using the Saccharomyces Genome Database (Stanford Genomic Resources) were obtained from Bioserve Biotechnologies. Since the HM loci and sequences distal to them are dispensable for growth, one primer (Omega3 [5'-CCTCGCACTA TCGCTGTTAT ACATGATGTC CCCAAAGCGT GTACAAATAA TTTTGTAGTA TTGTATCGGT AATATCATACA CAGAGCAGAT TGTACTGAGA GTGCACC-3']) contained yeast sequences (nonitalicized) between HMR and the next open reading frame (ORF) (omega 3) proximal to the centromere. Another primer (CHA1 [5'-GTAAGCATCA ACATATCCAA AACGTTGACA TATTTCTAGG CCGGCAATGC ACAGAATTTG TATAAAGGGG GACATGCTGCAG CGCATCTGTG CGGTATTTCA CACCGC-3']) contained yeast sequences (nonitalicized) between HML and the next ORF (CHA1) proximal to the centromere. Both primers also contained sequences (italics) that flanked the G418^r marker in plasmid pFA6a. Following PCR amplification of the G418^r marker from plasmid pFA6a, the LS20 strain was transformed with the PCR product and G418-resistant colonies were selected as described above. Circularization of chromosome III and retention of the u8 YAC were confirmed by transverse alternating field electrophoresis (TAFE) gel analysis as previously described (8, 9). Since circular chromosomes or YACs are unable to enter TAFE gels, the absence of chromosome III from the normal migration position of ~360 kb confirmed circularization in the G418-resistant colonies.

Genomic deletions of SIR2, SIR3, and SIR4 The SIR2, SIR3, and SIR4 loci were deleted in two LS20-derived strains containing the u8 YAC. These two LS20 strains lacked the genomic HO-cut sites either by sequential deletion of the YZ sites within HML and HMR or through a single-step-PCR-mediated complete deletion of HML and HMR that resulted in the circularization of chromosome III (see above). SIR2, SIR3, and SIR4 were deleted using the disruption plasmids pJH103.1, pDP91 (derived from pJH107.1), and pDM610.23, respectively (28). Plasmids pJH103.1 and pDP91were digested with the restriction endonucleases HindIII and EcoRI, respectively. Plasmid pDM610.23 was digested with the restriction endonucleases PvuII and PspAI. Restriction endonuclease-digested disruption plasmids were reintroduced into the appropriate host strains by transformation (66), and colonies were selected on leucine-deficient medium. Deletion of SIR2, SIR3, and SIR4 resulted in a nonmating phenotype in the LS20 strain that still retained the a1 and 2 transcription regulators within the HM loci (those deleted sequentially at each YZ junction). Deletions in this strain were confirmed by mating to the appropriate MATa and MAT tester strains as well as by Southern blot analysis (data not shown). Deletions of SIR2, SIR3, and SIR4 loci in the LS20 strain containing circular chromosome III were confirmed by Southern blot analysis alone.

Genomic deletions of RAD9 and RAD50. The RAD9 locus was deleted in a Sir⁺ strain using a one-step-PCR-mediated procedure (see above). Subsequently, SIR4 deletions were made in the rad9 strain using plasmid pDM610.23 as described above. The RAD50 locus was also deleted from Sir⁺ and sir4 strains using a one-step-PCR-mediated procedure. The following primers contain yeast sequences that flank the RAD9 and RAD50 loci, as well as sequences (italicized) that flank the hygromycin resistance gene within plasmid pAG32 (19): RAD9-LG (5'-CGTGGATATT TGCAACGATG AGCAATGTGA AGTGACCAAGATA GAGAAACGCC ATGTGACTGT CGCCCGTACA TT-3'), RAD9-RG (5'-CCAATCTTGA ACATTAACCA CTCCTGGCGT GTGGGAGGAT GTTCTTAGAC T GACAAGTTCT TGAAAACAAG AATC-3'), gr50c (5'-GCATGAGCGC TATCTATAAA TTATCTATTC AGGGCATACG GTCTT CGTACGCTG CAGGTCGAC-3'), and gr50d (5'-CGCAGTCTTA TAGGAGAGCT CCGTTTCTTC CAGGACATCA TTATA ATCGATGAAT TCGAGCTCG-3').

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Multiple copies of the silent mating locus HMR enhance indirect lethality resulting from a DSB. To identify genetic factors that influence the cellular response to a DSB, we screened a high-copy-number yeast genomic library for factors that would reduce the growth of an LS20 strain following induction of a single, HO-induced, unrepairable DSB at a YZ junction in a centromere-based plasmid (YZ-CEN). We chose this strain background because previously it had been shown that the in vivo induction of a nonrepairable DSB at a YZ site in a dispensable lambda DNA-based YAC (VS8) or chromosome did not lead to lethality (65, 67). However, a persistent DSB in YZ-CEN or a YZ YAC could lead to prolonged cell cycle delay at G₂ and indirect lethality in other strain backgrounds (CBY and NR85 [7, 8, 9]). We first established that the same persistent HO-induced DSB in the YZ-CEN plasmid or the YZ YACs did not cause lethality in the LS20 strain (Table 2). Contrary to results with the CBY and NR85 strains, the unrepairable break did not reduce the viability of LS20, indicating that the strain background may markedly affect the cellular response to an unrepaired DSB.

Depletion of Sir silencing complexes enhances indirect lethality. Plasmids containing transcriptionally inactive (silenced) mating type loci have been shown to require SIR silencing for plasmid replication and segregation (33), suggesting that high-copy-number plasmid expression may titrate SIR protein complexes away from other genomic sites. The results obtained with the high-copy-number HMR plasmid may therefore be due to (i) induction of additional DSBs at the YZ junction within HM loci in the plasmid or in the genome, (ii) a direct role of silencing proteins at the site of the DSB, or (iii) expression of MAT-controlled genes if the silent HML or HMR locus is activated by titration of Sir complexes.

A persistent DSB causes indirect lethality in sir2, sir3, and sir4 mutants. Since the SIR4 gene product appeared to play an important role in the cellular response to a DSB, we deleted each of the three genes required for telomere and mating type silencing (SIR2, SIR3, and SIR4). The Sir proteins silence the HML and HMR loci so that transcription of the mating type regulatory genes are repressed and the access of HO protein to the YZ target site is greatly inhibited (23, 37). Deletion of any of these genes did not affect growth rates of the LS20 cells (data not shown) but did eliminate silencing (data not shown). We also deleted the HO endonuclease target sites within the chromosomal silent mating loci HMR and HML at positions closely flanking the YZ junction. Therefore, the disruptions of the SIR genes could be examined for their effects on transcriptional silencing at HML and HMR versus their potential for inducing a DSB at these sites. The inability to make functional silencing complexes changed LS20 from an "a-like faker" phenotype (MAT had already been deleted [65]) into a nonmating, a1/2 haploid phenotype (HM⁺ in the sir2, sir3, and sir4 strains [data not shown]). As shown in Fig. 1, indirect lethality was observed only in sir2 , sir3, and sir4 strains containing the YZ YAC. Sir⁺ strains were unaffected by the DSB. These results demonstrate that the Sir protein complex plays a key role in the events proceeding from the appearance of an unrepairable DSB to indirect lethality.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Effects of a single persistent double-strand break within a dispensable YAC on survival. LS20 (with the MAT locus deleted) was transformed with the human artificial chromosome u8, which contains a YZ-URA3 sequence embedded in a cluster of ALU repeats 70 kb from the telomere. Either the HO target sites within the HML and HMR loci were precisely deleted (HM+) or HML and HMR were completely deleted (hm by circularizing chromosome III in LS20 strains containing the u8 YAC. SIR2, SIR3, SIR4, RAD50, and both SIR4 and RAD50 were subsequently deleted from HM+ and hm strains. RAD9 was deleted from a Sir+ HM+ strain, and SIR4 was subsequently deleted. A Sir+ HM+ strain expressing the a1 and 2 transcriptional regulators from pCB115 is denoted a1/2 HM+. Cells were grown to logarithmic phase (1 × 107 to 3 × 107 cells/ml) in liquid SC-GLU medium (88) lacking URA to maintain selection for the YAC. In the case of the strain containing plasmid pCB115, the medium also lacked leucine to maintain plasmid selection. Cells were washed in sugarless medium, and appropriate dilutions were plated to SC-GLU and SC-GAL plates (with URA to remove selection for the YAC). Survival was determined by relative colony-forming abilities on GAL versus GLU plates after 4 days of incubation at 30°C. Each small filled circle represents the survival data from one experiment. Large open circles and crosses denote the mean survival ± 1 standard deviation for each strain.

SIR silencing of mating type provides toleration of a persistent DSB. The toleration of a DSB in Sir⁺ strains might be due to the role that Sir proteins have on chromatin structure in the vicinity of the DSB site or alternatively from the repression of mating type regulatory genes within HML and HMR loci by Sir complexes. To address this issue we deleted HML and HMR (these strains are denoted hm) using a one-step-PCR-mediated gene disruption that resulted in a circular chromosome III [see Materials and Methods]). Following circularization of chromosome III, the SIR2, SIR3, and SIR4 genes were individually deleted. As expected, the circularization of chromosome III had no effect on the growth of LS20 since naturally occurring circular derivatives have been previously shown to be stable during replication and segregation (72). As shown in Fig. 1, indirect lethality was not observed in any of these strains following induction of the persistent DSB. These results demonstrate that HML and HMR are required for indirect lethality to occur when Sir2, Sir3, and Sir4 products are absent. In the absence of silencing, HMR and HML normally produce the a1-2 repressor complex that confers an a1/2 haploid (nonmating) phenotype upon the LS20 strain. It therefore appears that a gene(s) under mating type regulation is responsible for the observed DSB responses.

Commitment to death in sir4 mutants occurs later than an HO-induced DSB in a dispensable YAC. Since a DSB can initiate lethality in the Sir mutants, we wanted to establish if the lethality is reversible and, if so, when the lethal effects of the break are irreversible. We used the previously described pullback approach (8) in which logarithmically growing cells are embedded in GAL-URA medium to induce the HO endonuclease and at various times the plates are overlaid with GLU-URA to shut off HO. Since the HO cut site in the YAC is closely linked to the URA3 marker and the break is not repaired, it is possible to genetically determine when the break occurs. Similarly, cells are plated within GAL+URA medium and overlaid with GLU+URA medium to determine when cells cannot be rescued and become committed to death.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Commitment to death in the sir4 HM+ strain following induction of a persistent DSB within the u8 YAC. A pullback procedure (8) was used where sir4 (triangles) or Sir+ (circles) HM+ strains containing the u8 YAC were plated within GAL medium with URA (filled symbols) or without URA (open symbols) to induce expression of the HO endonuclease. At the indicated times, the plates were overlaid with either GLU medium with URA or GLU medium without URA to repress expression of the HO endonuclease. Decreased survival on plates with GAL medium lacking URA requires HO-induced cutting of the YAC and concomitant loss of the URA3 marker from DNA degradation. Each data point is the average of results from two to three experiments. Results with the sir4 rad50 strain and the sir4 strain were comparable (data not shown).

Adaptation to DSB-induced G₂ arrest is decreased in Sir mutants. To address the role of mating type on the ability of a DSB to trigger cell cycle arrest, we monitored microscopically wild-type, sir2 , sir3, and sir4 cells that either contained HML and HMR (with HO sites deleted but a1 and 2 sites intact) or completely lacked HML and HMR (the strains contained circular chromosome III). Since the HML and HMR loci are derepressed in a sir background, the HML HMR cells expressed the a1 and 2 repressors and therefore appeared as an a1/2 (nonmating) haploid, while the hml hmr strain retained an a-like faker haploid mating type phenotype.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Effects of a single persistent double-strand break within a dispensable YAC on cell cycle progression of single (G1) cells. Either the HO target sites in sir2 , sir3 , sir4, and rad50 LS20-derived strains containing the u8 YAC were precisely deleted (HM+) (A) or HML and HMR were completely deleted (hm) (B). The HM+ cells retained the a1/2 mating type gene regulators and expressed an a1/2 haploid (nonmating) phenotype in sir strains. Cells were grown and washed as described in the legend to Fig. 1 and then spread onto SC-GAL plates using a multipin pronging device. For each mutant strain, two isolates were examined microscopically using a Singer MSM dissecting microscope and compared to the corresponding wild-type parental strain (8, 9). Individual cells (>400 for each bar in the graph) were examined immediately after being plated to the SC-GAL medium and 12 h later. At the time of plating, all isolates had similar distributions of cells: ~50% were G1 (single cells), ~25% were S (small budded), and ~25% were G2 (large budded). Presented are the results for the cells that were in G1 (unbudded cells) (A and B) at the time of plating. G1 cells either remained as initially plated (no progress), progressed to the budded stage, progressed to the 3- to 4-cell stage, or formed microcolonies (5 to 10 cells). The mean percentage of cells for the wild-type parental strain was subtracted from the mean percentage obtained for each mutant strain in each category. Mutant strains that contained a higher percentage of cells in a given category had positive values, whereas mutant strains that had fewer cells in a given category had negative values. The deviations from the wild-type value (baseline = 0) for these mean percentages are indicated by bars. An * over a bar indicates a deviation from the wild-type value that is significant (±1 standard deviation). Cells that were in G2 at the time of plating either remained as initially plated (budded), progressed to the 3- to 4-cell stage, or formed microcolonies (5 to 10 cells and >10 cells; data not shown). For the sir2, sir3, and sir4 cells able to form an a1/2 haploid phenotype (HM+) (A), a persistent DSB in the u8 YAC leads to the accumulation of cells at G2 (i.e., as budded cells [22, 84]). No G2 delay was observed in sir2, sir3, and sir4 cells lacking the u8 YAC or those without the HML and HMR loci (hm) (B). Similarly, sir2, sir3, and sir4 cells able to form an a1/2 haploid phenotype that were in S or G2 (data not shown) at the time of plating, also accumulated at the subsequent G2 stage (i.e., as budded cells [22, 84]).

View larger version (273K): [in this window] [in a new window]   FIG. 4.   Delayed cell cycle progression of sir4 cells following induction of a persistent DSB in a dispensable YAC. Cells were grown as described in the legend to Fig. 1. (A) Single (G1) cells of SIR4 (circles), sir4 (triangles), rad9 sir4 (squares), and sir4 rad50 (diamonds) strains were micromanipulated into a grid pattern on an SC-GAL plate and photographed at hourly intervals. Cells with RAD9 deleted show no G2 delay. Cells were scored as percentages of cells that had progressed past the G2/M checkpoint and formed a microcolony containing three or more cells. Zero hour is the time of initial plating. (B) Representative photomicrographs at 12 h (left panels) and 24 h (right panels) for the SIR4 (upper panels) and sir4 (lower panels) HM+ cells are depicted. Cell cycle progression is delayed in the sir4 strain as the result of a more prolonged arrest in the initial and second (arrows) G2 phases. This is followed by repeated cycles of G2 arrest, adaptation, and rearrest in G2 (see the text). (C) Representative photomicrographs at 12 h (left panels) and 24 h (right panels) for the sir4 rad50 HM+ (upper panels), sir4 rad50 hm (middle panels), and sir4 rad50 HM+ cells without a persistent DSB (no YAC in lower panels) are depicted. Cell cycle arrest in the sir4 rad50 strain is specific for a YAC DSB and requires HML and HMR.

Prolonged G₂ arrest in sir4 a1/2 haploid cells is dependent on RAD9. To further characterize the response of various mutants to a DSB in terms of time of onset of cell division, time in G₂, ability to adapt or rearrest after the first division, and dependence on the RAD9 checkpoint gene, individual cells were monitored microscopically at hourly intervals for 36 h. Cells of each strain were micromanipulated onto a GAL plate in a grid pattern within one microscopic field of view. These were photographed at hourly intervals, and the mean times for the onset of cell division, as well as the time spent as budded cells or as four-cell microcolonies, were calculated. Depicted in Fig. 4A are the time courses for the formation of microcolonies (at least three cells) of SIR4, sir4, rad9 sir4, and sir4 rad50 HM⁺ cells after single (G₁) cells were plated to GAL. The times of onset of initial bud formation in all the strains examined were similar (~6 h) (data not shown). Overall, the SIR4 cells were delayed in G₂ for less time and had formed a higher percentage of large microcolonies (more than four cells; 70%) than the sir4 cells (20%) after 24 h (Fig. 4B). Half (50%) of the sir4 cells were still in the large-budded stage or had rearrested as a chain of four cells after 24 h of growth on GAL. These arrested sir4 cells could go on to form microcolonies of ~50 to ~100 nonviable cells, which did not progress further. Unlike wild-type cells, the individual sir cells appeared to exhibit irregular growth patterns within the microcolonies, which would explain their distorted shapes (Fig. 4B). These microcolonies account for the increased lethality seen in this strain compared to the lethality of SIR4 cells (the relative plating efficiencies on GLU versus GAL in this experiment were 0.46 and 1.1 for the sir4 and SIR4 strains, respectively).

The RAD50-dependent end-joining repair pathway is not required for toleration of a persistent DSB. In some strain backgrounds the SIR gene products appear to play a direct role in NHEJ repair of DSBs. In addition to the Sir proteins, Yku70 or Yku80 and the Rad50-Mre11-Xrs2 complex are all required for NHEJ repair. Furthermore, deletion of RAD50 suppressed permanent arrest in a yku70 mutant by enhancing adaptation to an HO-induced, chromosomal DSB (38). We therefore deleted RAD50 to determine if the observed effects of SIR on cell cycle adaptation and toleration of a DSB were mediated through this pathway.

Absence of Sir4 does not increase sensitivity to -ray or MMS-induced damage. Recently it was proposed that the SIR4 gene might play a direct role in the repair of radiation-induced breaks (11, 81) or MMS-induced damage (48) via NHEJ. The sensitivity of rad52 cells, which are unable to undergo homologous recombinational repair of DSBs, was increased by a sir4 mutation (81). However, little effect could be discerned for the sir4 mutation in haploid Rad⁺ cells, which is surprising since haploid G₁ cells are radiation sensitive due to a lack of opportunities for recombinational repair (12). We, therefore, examined the radiation sensitivity of stationary-phase cells (~95% of cells in G₁) (Fig. 5A) and the MMS sensitivity of logarithmically growing cells (Fig. 5B) with various combinations of SIR, sir4, and RAD50 alleles.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Survival following -irradiation (A) or MMS treatment (B) of haploid Sir+ (circles), sir4 (triangles), rad50 (squares), and sir4 rad50 (diamonds) cells. (A) For -irradiation, the LS20-derived strains were grown to stationary phase in YEPD. Based on cell appearance, ~95% of the cells were in G1 (i.e., unbudded) at the time of irradiation. Survival was determined by counting relative numbers of CFU after 3 days of growth at 30°C on YEPD plates. (B) For MMS treatment, survival was determined following treatment of logarithmically growing Sir+ (circles), sir4 (triangles), rad50 (squares), and sir4 rad50 (diamonds) cells. Cells in SC-GLU medium were treated with 0.01% (filled symbols) or 0.03% (open symbols) MMS and reincubated with shaking at 30°C. At the indicated times, cells were diluted in water and plated onto YEPD plates. Survival was determined by counting relative numbers of colonies after 4 days of growth at 30°C. Each data point is the mean of results of two to three replica experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In addition to the elaborate systems involved in the repair of DNA damage, there are many genes that influence cell progression in response to chromosomal lesions. Checkpoint genes such as RAD9 serve to arrest cell cycle progression at G₂ in the presence of persistent damage. Presumably, this allows additional time for repair (84). However, under conditions where a DSB remains unrepaired in an LS20 strain, either because of a defect in the DSB recombinational repair pathway (74) or lack of a homologue (65, 67), the arrest is only temporary. The reentry of cells into the cell cycle in the presence of unrepaired damage has been termed adaptation (20, 58, 83), and factors that are responsible for the adaptation response to an unrepaired chromosomal DSB in a rad52 mutant have been identified (78). We sought to identify additional factors that play a role in adaptation and lethality using a system where a persistent DSB is produced in a dispensable YAC.

An indirect role for SIR2, SIR3, and SIR4 in cell cycle adaptation and toleration of a DSB. We found that SIR genes play an indirect role in adaptation. The approach that we took was motivated by our observations that an unrepairable DSB in some repair-proficient strain backgrounds (Rad⁺) leads to extended G₂ arrest and indirect lethality but that there is only a modest delay and no lethality in other strains (references 65 and 67 and this report). An unrepairable DSB in the repair-proficient LS20 strain normally leads to several hours of delay in G₂ (~6 h) (Fig. 4A) and is well tolerated since it does not affect survival.

SIR4 does not play a direct role in the repair of -ray- or MMS-induced damage. The SIR gene products are part of a multiprotein complex that determines chromatin structure and enables transcriptional silencing of genes at both silent mating type loci and telomeres. In addition to silencing, Sir4 also has a key role in other aspects of DNA metabolism. Sir4 is required for mitotic chromosomal stability and efficient segregation of unstable plasmids (3, 57, 33). Recently, it was shown that Sir4 physically interacts with Hdf1 (now designated Yku70), a yeast homologue of Ku70 required for end-joining repair of DSBs (81). Furthermore, using plasmid end-joining repair assays, SIR2, SIR3, and SIR4 have all been shown to be essential for Ku-dependent DSB DNA repair (11).

rad50

Deletion of RAD50 does not enhance adaptation to a DSB in sir4 strains. A number of proteins, including Hdf1, Mre11, Rad1, Rad5, Rad9, Rad17, Rad24, Rad50, Xrs2, and Mec3, have been reported to bind to or interacting with the exposed ends of a DSB in yeast (1, 16, 44, 51, 81). Recently, Yku70 or Yku80, Mre11, and Rad50 were shown to function as a complex in both Ku-dependent NHEJ repair and telomere length maintenance (10, 11). In addition, both Rad50 and Mre11 are required for the initiation of spontaneous breaks in meiosis resulting in a reduction in meiotic recombination (52). Furthermore, Mre11, Rad50, and Xrs2 have been shown to interact as a complex. While null mutations are proficient in mating type switching and spontaneous mitotic recombination, they are sensitive to the killing effects of ionizing radiation (18, 27, 45, 46, 55) or restriction enzyme-induced DSBs (40). Deletion of RAD50, MRE11, or XRS2 resulted in decreased 5'-to-3' degradation at HO-induced DSBs and also greatly reduced the level of NHEJ repair of this lesion (51, 74). Cells with YKU70 deleted expressed a permanent G₂ arrest following induction of a single HO-induced chromosomal break, and this was attributed to enhanced 5'-3' degradation at the break site (38). Deletion of either RAD50 or MRE11 in the yku70 strain enabled the cells to adapt to the DSB and resume cell cycle progression due to reduced degradation at the break site (38). In our system, deletion of RAD50 did not rescue logarithmically growing sir4 HM⁺ cells from repeated cell cycle arrests. Thus, it is unlikely that, in the LS20 strain background, the prolonged G₂ arrest and lethality in sir4 strains is due to greatly enhanced 5'-3' recision at the DSB site.

Mating type control of the repair of DSBs. It has been suggested that the Ku, Sir2, Sir3, and Sir4 proteins are required for both DSB repair and silencing at telomeres (11). Furthermore, other studies have described a redistribution of Sir proteins from telomere regions to DSB damage sites that is dependent on MEC1 and RAD9 (48, 49, 50). However, recent studies suggest that the effects of SIR4 deletions may be mostly indirect, resulting from derepression of the silent mating loci and formation of an a1/2 haploid phenotype (5, 39). These studies suggest that another gene(s) under mating type control may be involved in DSB repair. Therefore, a haploid-specific gene(s) may be turned off; alternatively, a diploid-specific gene(s) may be turned on.

Implications of SIR4-dependent toleration of DNA damage. A response similar to that in sir cells was found in the diploid CBY strain in that it also shows poor adaptation and prolonged G₂ arrest following the appearance of an unrepaired DSB (8). The Sir4 silencing system may be part of an adaptive response that enables haploid cells to tolerate unrepaired DNA damage. Only in the absence of both SIR4 and the ability to express a1 and 2 is the lesion rendered lethal by virtue of delayed adaptation resulting in cell death. The redundant nature of this damage toleration system ensures that the adverse genetic consequences of an unrepaired break will be avoided.