- Open Access
Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability
Genome Integrity volume 2, Article number: 8 (2011)
Genome instability is associated with human cancers and chromosome breakage syndromes, including Bloom's syndrome, caused by inactivation of BLM helicase. Numerous mutations that lead to genome instability are known, yet how they interact genetically is poorly understood.
We show that spontaneous translocations that arise by nonallelic homologous recombination in DNA-damage-checkpoint-defective yeast lacking the BLM-related Sgs1 helicase (sgs1Δ mec3Δ) are inhibited if cells lack Mec1/ATR kinase. Tel1/ATM, in contrast, acts as a suppressor independently of Mec3 and Sgs1. Translocations are also inhibited in cells lacking Dun1 kinase, but not in cells defective in a parallel checkpoint branch defined by Chk1 kinase. While we had previously shown that RAD51 deletion did not inhibit translocation formation, RAD59 deletion led to inhibition comparable to the rad52Δ mutation. A candidate screen of other DNA metabolic factors identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1Δ mutant, becoming even more important for chromosomal stability upon MEC3 deletion. We determined that the C-terminal third of Exo1, harboring mismatch repair protein binding sites and phosphorylation sites, is dispensable for Exo1's roles in chromosomal rearrangement suppression, mutation avoidance and resistance to DNA-damaging agents.
Our findings suggest that translocations between related genes can form by Rad59-dependent, Rad51-independent homologous recombination, which is independently suppressed by Sgs1, Tel1, Mec3 and Exo1 but promoted by Dun1 and the telomerase-inhibitor Mec1. We propose a model for the functional interaction between mitotic recombination and the DNA-damage checkpoint in the suppression of chromosomal rearrangements in sgs1Δ cells.
Eukaryotic cells have mechanisms at their disposal for the detection and repair of spontaneous and induced DNA lesions, thus preventing them from giving rise to potentially abnormal daughter cells. However, if these mechanisms are defective or overwhelmed by damage, deleterious chromosomal rearrangements can arise. A multitude of genes and genetic pathways for the maintenance of genome stability has been identified mostly using genetic screens in simple model organisms such as the yeast Saccharomyces cerevisiae. They include DNA damage checkpoints, DNA repair factors and proteins for processing of recombination substrates and intermediates [1–10]. The importance of the same mechanisms for maintaining genome stability in human cells is highlighted by the association of mutations in the human homologues of these yeast genes with chromosome breakage syndromes, which are characterized by signs of premature aging and/or cancer development. The syndromes include Nijmegen breakage syndrome associated with mutations in NBS1, the homologue of yeast XRS2 [11–13]; Bloom's syndrome and Werner syndrome associated with mutations in BLM and WRN , respectively, both related to yeast SGS1 [14, 15]; and ataxia telangiectasia associated with mutations in ATM , which is related to yeast TEL1 .
Yeast SGS1 encodes a 5' to 3' DNA helicase that preferentially unwinds three- and four-way junctions typical of replication and recombination intermediates and has recently been shown to collaborate with Exo1 in the long-range processing of double-strand breaks (DBSs) [18–21]. Without Sgs1, cells accumulate gross-chromosomal rearrangements (GCRs), exhibit elevated levels of mitotic recombination, have a reduced replicative lifespan and are sensitive to chemicals that alkylate DNA or slow replication forks [2, 22–26]. Among DNA-damage checkpoint components, Mec1 kinase, also considered the homolog of mammalian ATR [27–29], has been identified as one of the strongest suppressors of GCRs in yeast [3, 4]. Other cellular phenotypes of mec1Δ mutants include increased sensitivity to DNA damaging agents and deficient DNA-damage checkpoint response , instability of stalled forks , accumulation of DNA breaks  and, in addition to these mitotic defects, deficiencies in meiotic checkpoint activation and recombination [33–35]. In contrast to Mec1, cells lacking the Tel1 checkpoint kinase, which is related to mammalian ATM [17, 36], are not sensitive to DNA damaging agents , do not accumulate GCRs above wildtype levels , but show telomere erosion . Synergistic interactions between mec1Δ and tel1Δ mutations have been reported for many phenotypes, suggesting a functional relationship and redundancy between the two kinases [3, 17, 37, 38]. Other checkpoint components, such as those involved in sensing DNA damage (Mec3, Rad24), appear to have only small to moderate roles in suppressing GCRs in yeast [3, 4]. In cells lacking the Sgs1 helicase, however, Mec3 and Rad24 strongly suppress overall genome instability [3, 4] as well as the formation of spontaneous, recurring translocations between short identical sequences in non-allelic, but related, DNA sequences . Utilizing the high susceptibility of the sgs1Δ mec3Δ mutant to recurring translocation formation between CAN1, LYP1 and ALP1, we have in the current study conducted a candidate screen to identify two types of DNA metabolic factors - those that are required for the formation of recurring translocations in the sgs1Δ mec3Δ mutant and those that act independently of Sgs1 and Mec3 to suppress translocations. For this purpose, mec1Δ, tel1Δ, dun1Δ, chk1Δ and rad59Δ mutations were introduced into the sgs1Δ mec3Δ mutant and the accumulation of recurring translocations was assessed. We further determined how the lack of other DNA metabolic factors (yen1Δ, lig4Δ, exo1Δ, rad1Δ, pol32Δ) affects the accumulation of genome rearrangements, identifying a strong synergistic interaction between sgs1Δ and exo1Δ. We propose an integrated model for independent, functional interactions between Sgs1, HR subpathways and various DNA-damage-checkpoint branches in the suppression of chromosomal rearrangements.
Results and discussion
Functional interaction between Sgs1 and DNA-damage checkpoint components Mec3, Mec1, Tel1, Dun1 and Chk1 in the suppression of chromosomal translocations
Chromosomal translocations between short stretches of homology in nonallelic sequences that are naturally present in the yeast genome, such as the highly similar, but diverged CAN1 (on chromosome V), ALP1 and LYP1 genes (on chromosome XIV, 60-65% identity) , are normally suppressed in yeast. However, they are recurrent in sgs1Δ mutants with certain additional DNA-metabolic defects, including mec3Δ, rad24Δ, cac1Δ, asf1Δ and rfc5-1 . One of the mutants most susceptible to recurring translocations between the CAN1, LYP1 and ALP1 loci is the sgs1Δ mec3Δ mutant, whereas translocations are not found in the sgs1Δ mec1Δ mutant . Here, we wanted to test whether the lack of CAN1/LYP1/ALP1 translocations in the sgs1Δ mec1Δ mutant meant that Mec1 was not a suppressor of translocations and therefore its deletion had no affect on translocation formation, or that Mec1 was actually required for the formation of viable chromosomal translocations. If the latter was true, we expected that introducing a mec1Δ mutation into the highly susceptible sgs1Δ mec3Δ strain should inhibit the accumulation CAN1/LYP1/ALP1 translocations. Indeed, we found that while deleting MEC1 led to a synergistic increase (~ 7-fold) in the rate of all GCR types compared to the sgs1Δ mec3Δ mutant (P < 0.0001), screening of GCR clones obtained from 431 individual sgs1Δ mec3Δ mec1Δ cultures failed to reveal a single CAN1/LYP1/ALP1 translocation, signifying a > 7-fold decrease in the translocation rate compared to the sgs1Δ mec3Δ mutant (Table 1). The synergistic GCR rate increase in the sgs1Δ mec3Δ mec1Δ mutant shows that Mec1 can activate its targets through Mec3-independent sensing of DNA damage. This may occur by Mec1-Ddc2 itself recognizing and binding to DNA lesions [39, 40] or through DNA-damage sensors other than the Mec3 clamp signaling to Mec1. The synergistic GCR rate increase in the sgs1Δ mec3Δ mec1Δ mutant also indicates that the failure to form CAN1/LYP1/ALP1 translocations when MEC1 is deleted is not due to an inability to form viable GCRs, but rather suggests that DNA lesions are channeled into GCR pathways other than homology-driven translocation. Most likely, Mec1 promotes chromosomal translocations by inhibiting de novo telomere synthesis at chromosome breaks , for example by phosphorylating the telomerase-inhibitor Pif1  and by phosphorylating Cdc13 and thus preventing its accumulation at DNA breaks . In a haploid wildtype cell, these chromosomal translocations are expected to be rare due to restraints placed on homologous recombination events by the need for relative long regions of sequence identity. However, when the restraints on homologous recombination are relaxed and spontaneous DNA lesions are not properly detected by the DNA-damage checkpoint, as could be assumed for the sgs1Δ mec3Δ mutant, chromosomal translocations are promoted and occur between much shorter regions of sequence identity, such as the 5-41-bp segments present in CAN1, LYP1 and ALP1.
Deleting TEL1, which encodes another DNA-damage checkpoint kinase that is considered at least partially functionally redundant with Mec1, had the same effect as deleting MEC1 on the accumulation of all types of GCR (Table 1), as evidenced by the 44-fold increase in the overall GCR rate compared to the sgs1Δ mec3Δ mutant (5.7 × 10-6 versus 1.3 × 10-7, P < 0.0001). However, deleting TEL1 had the opposite effect on CAN1/LYP1/ALP1 translocation formation (Table 1). Instead of inhibiting CAN1/LYP1/ALP1 translocations like the mec1Δ mutation, the tel1Δ mutation led to an increase (~15-fold) in CAN1/LYP1/ALP1 translocations (Table 1). Unlike mec1Δ mutants, mutants lacking Tel1 are impaired in their ability to maintain telomeres  and may thus be unable to heal DNA breaks by de novo telomere addition. Thus, in the absence of Tel1, DNA breaks may be channeled into alternative pathways for repair, such as HR, and more frequently give rise to CAN1/LYP1/ALP1 rearrangements under conditions that favor aberrant HR such as those in the sgs1Δ mec3Δ mutant. That failure to activate either Tel1 or Mec3-checkpoint pathways contributes independently to recurrent CAN1/LYP1/ALP1 translocation formation suggests that both ssDNA overhangs or gaps, thought to be sensed in a Mec3-dependent manner, and DSBs, thought to be sensed in a Tel1-dependent manner, can lead to CAN1/LYP1/ALP1 translocations and that they accumulate in unperturbed sgs1Δ cells spontaneously. The synergistic increase in overall genome instability in the sgs1Δ mec3Δ tel1Δ mutant might also indicate that in the absence of lesion binding by the Mec3 clamp some lesions are further processed and eventually detected by the Tel1-dependent pathway. For example, a stalled replication fork might eventually be processed into double-stranded ends in an attempt at fork restart by fork regression or template-switching.
Thus, both Tel1 and Mec1 act independently of Mec3 and Sgs1 to strongly suppress overall genome instability, but they affect CAN1/LYP1/ALP1 translocation formation in opposite ways. The inhibition of CAN1/LYP1/ALP1 translocations upon MEC1 deletion as opposed to their increase upon TEL1 deletion can most likely be explained by their opposite effects on telomere synthesis, with Mec1 inhibiting it and Tel1 promoting it. This is also consistent with the previous report of different GCR spectra in the tel1Δ and mec1Δ single mutants . Apart from regulating telomere maintenance factors, it is also conceivable that the DNA-damage checkpoint-dependent phosphorylation of homologous recombination factors, such as Rad55, Slx4 and Mus81 [43–47] contributes to differential regulation of translocation formation in the sgs1Δ mec3Δ mutant.
The opposing effects of Tel1 and Mec1 on CAN1/LYP1/ALP1 translocation formation led us to investigate other DNA-damage checkpoint components in sgs1Δ and sgs1Δ mec3Δ mutants. We found that deletion of either CHK1 or DUN1 led to a synergistic increase in overall genome instability when combined with an sgs1Δ mutation (P < 0.0001), however only the dun1Δ mutation caused a further significant GCR rate increase in the sgs1Δ mec3Δ mutant (P < 0.0001, Table 1) whereas the chk1Δ mutation did not (P = 0.1615, Table 1). Analysis of the GCR types revealed accumulation of CAN1/LYP1/ALP1 translocations in the Chk1-deficient sgs1Δ mec3Δ mutant at a similar rate as in the sgs1Δ mec3Δ mutant, but not in the Dun1-deficient sgs1Δ mec3Δ mutant (Table 1), indicating that Dun1, like Mec1, promotes translocation events between CAN1, LYP1 and ALP1 whereas Chk1 does not. This is likely due to Mec1-mediated activation of Dun1 kinase, which in turn inactivates the transcription repressor Crt1, thus allowing transcription of several DNA-damage inducible genes [48, 49]. Chk1 kinase is also activated through Mec1 in response to DNA damage and causes a transient G2/M arrest by blocking anaphase progression [50, 51]. However, in contrast to Dun1, Chk1 is not thought to regulate DNA repair pathways, and its deletion did not inhibit translocation formation in the sgs1Δ mec3Δ mutant (Table 1). As expected, deletion of RAD17, which encodes another subunit of the Mec3/Rad17/Ddc1 checkpoint clamp, had a similar effect on CAN1/LYP1/ALP1 translocation formation in the sgs1Δ tel1Δ mutant as deletion of MEC3 (Table 1). The detection of a CAN1/LYP1/ALP1 translocation in two strains that expressed wildtype Sgs1 (mec3Δ tel1Δ (Table 1) and mec3Δ tel1Δ rad17Δ (not shown)) suggests that even in the presence of wildtype Sgs1 cells may accumulate CAN1/LYP1/ALP1 translocations as long as they are deficient in at least two independent suppressors of translocation formation, such as Tel1 and Mec3 identified here.
Deletion of RAD59inhibits spontaneous interchromosomal translocations between short repeats
We previously showed that translocations between CAN1, LYP1 and ALP1 in the sgs1Δ mec3Δ mutant are Rad52-dependent, but translocations still formed when Rad51 was absent . To assess the role of other HR factors in translocation formation we deleted RAD59 in the highly susceptible sgs1Δ mec3Δ mutant and measured the rate of accumulating all types of GCRs as well as CAN1/LYP1/ALP1 translocations. One CAN1/LYP1/ALP1 rearrangement was identified among GCR clones obtained from 158 independent cultures of the sgs1Δ mec3Δ rad59Δ mutant (Table 2), indicating a 10-fold reduction in the CAN1/LYP1/ALP1 translocation rate compared to the sgs1Δ mec3Δ mutant. Thus, similar to Rad52, Rad59 is required for interchromosomal translocations between short identical sequences in related genes. If Rad59 was indeed required for translocation formation, we predicted that the formation of CAN1/LYP1/ALP1 translocations in the sgs1Δ mec3Δ rad51Δ mutant would also be inhibited by a rad59Δ mutation. Thus, we generated an sgs1Δ mec3Δ rad51Δ rad59Δ mutant and screened for CAN1/LYP1/ALP1 translocations. Among 168 independent GCR clones we identified one CAN1/LYP1/ALP1 translocation, indicative of a 28-fold reduction of the translocation rate compared to the sgs1Δ mec3Δ rad51Δ mutant (Table 2). Thus translocations between CAN1, LYP1 and/or ALP1 can form through Rad52/Rad59-mediated HR that does not require Rad51. Rad59 has recently also been shown to contribute to GCRs mediated by certain Ty-elements and to translocations involving short DNA sequences of limited sequence identity [6, 52].
While Rad52 is required for all HR in yeast, some DNA breaks can be repaired by HR pathways that do not require Rad51, including single-strand annealing (SSA), break-induced replication (BIR) and recombination-mediated telomere-lengthening Type II [53–58]. SSA is a mechanism for the repair of a DSB between repeated DNA elements and requires Rad59, but not Rad51 . In order for the interchromosomal CAN1/LYP1/ALP1 rearrangements to arise by SSA, however, at least two DSBs would have to occur in the same cell - one DSB within or downstream of CAN1 on chromosome V and one DSB near ALP1 (or LYP1) on chromosome XIV. Resection would expose the short stretches of homology shared by CAN1 and ALP1 (or LYP1) , allowing them to anneal, followed by removal of the nonhomologous overhangs and ligation. Rad59-dependent, Rad51-independent interchromosomal translocation between his3 fragments was recently shown after induction of HO-breaks in the two recombining chromosomes . Such an interchromosomal SSA event could also produce the types of rearrangements we have observed between CAN1, LYP1 and ALP1; however, the ends of chromosomes V and XIV not engaged in the SSA event would be left unrepaired and most likely would be lost after cell division unless the recombination event occurs in G2/M when sister-chromatids are present. Moreover, since we have shown that wildtype copies of LYP1 and ALP1 are still present in recombinants with CAN1/LYP1/ALP1 rearrangements, indicative of a nonreciprocal translocation event , and the parts of chromosome XIV that would be lost after SSA contain essential genes, SSA is unlikely to be the main recombination mechanism that gives rise to CAN1/LYP1/ALP1 rearrangements.
Besides SSA, BIR also matches the genetic requirements for CAN1/LYP1/ALP1 translocation formation. BIR is initiated by invasion of a duplex by a single-stranded 3'end of a one-sided DBS followed by replication to the chromosome end. Although Sgs1 has roles in recombination, specifically sister-chromatid exchange and resolution of recombination intermediates [2, 9, 62–64], it is not required for Rad51-independent BIR . In contrast to SSA, the nonreciprocal nature of BIR events would maintain an intact copy of chromosome XIV in addition to the chromosome V/XIV translocation, suggesting that it is the more likely mechanism involved in CAN1/LYP1/ALP1 translocation. BIR has been implicated in the repair of one-sided DSBs, such as replication forks that collapsed at a single-strand break. BIR is also thought to allow telomerase-deficient cells (tlc1Δ), whose telomeres have shortened to a point where cells can no longer proliferate, to survive by extending what could be considered a one-sided DSB. Survivors can arise either by adding subtelomeric Y' elements in a Rad51-dependent mechanism (Type I) or by adding telomeric (G1-3T)n repeats in a Rad51-independent, but Rad59-dependent mechanism (Type II) [53–55]. The differential requirement for Rad51 and Rad59 in these two pathways is thought to result from the differences in length and sequence identity of the recombination substrates for Type I and Type II . The long, nearly identical (~1% variation within the same strain) Y' elements  are thought to be better substrates for Rad51-mediated strand invasion, whereas Rad59 is able to use the shorter stretches of homology likely to be found within the highly variable (G1-3T)n repeats . Besides BIR, evidence of homology-length dependency is also seen in gene conversion, with Rad59 becoming increasingly important as the length of sequence homology decreases . This length-dependency may also explain our observation that CAN1/LYP1/ALP1 rearrangements, which show short regions of homology at the breakpoints [10, 60], are inhibited by deletion of RAD59, but not by deletion of RAD51. Despite this differential effect on chromosome rearrangements between CAN1, LYP1 and ALP1, we observed no difference in the rate of overall genome instability between sgs1Δ mec3Δ rad51Δ and sgs1Δ mec3Δ rad59Δ mutants (P = 0.6892, Table 2), suggesting that the DNA lesions that give rise to viable GCRs are accessible to multiple repair pathways.
Candidate screen reveals EXO1as a strong suppressor of GCR formation in cells lacking Sgs1
To assess the possible role of other DNA metabolic factors in the suppression or formation of GCRs in cells lacking Sgs1, we introduced exo1Δ, pol32Δ, rad1Δ, lig4Δ and yen1Δ mutations into sgs1Δ and sgs1Δ mec3Δ mutants. Screening of the single, double and triple mutants revealed that RAD1, POL32, LIG4 and YEN1 are not strong suppressors of GCRs in wildtype cells, or in sgs1Δ or sgs1Δ mec3Δ mutants (Table 3). However, when we assessed the formation of CAN1/LYP1/ALP1 translocations in sgs1Δ mec3Δ mutants with pol32Δ or rad1Δ mutations we found that in both triple mutants CAN1/LYP1/ALP1 translocations were inhibited, revealing one CAN1/LYP1 translocation among 98 independent GCR clones in the sgs1Δ mec3Δ pol32Δ mutant and none (0/55) in the sgs1Δ mec3Δ rad1Δ mutant. Pol32, a nonessential subunit of polymerase δ that promotes processivity of the polymerase, is not required for SSA, but for DNA repair processes that involve extensive DNA synthesis, such as BIR , consistent with BIR being a pathway for CAN1/LYP1/ALP1 translocation formation. Although Rad1, a subunit of the Rad1-Rad10 nuclease critical for the removal of nonhomologous overhangs from annealed single strands in processes such as SSA [67, 68], has not been shown to be required for BIR, it has been implicated in the removal of nonhomologous overhangs during GCR formation  and in recombination events that combine BIR and SSA processes [70, 71].
Deletion of EXO1, coding for a nuclease with 5' to 3' exonuclease and flap-endonuclease activities, which has roles in mitotic and meiotic recombination as well as mutation avoidance and is thought to cooperate with Sgs1 in the processing of DSBs [19, 72], induced the largest synergistic GCR rate increase we have observed to date in the sgs1Δ mutant. While sgs1Δ and exo1Δ single mutants exhibited moderately increased GCR rates compared to wildtype, the GCR rate of the sgs1Δ exo1Δ mutant was several hundred-fold higher than the rates of the single mutants (P < 0.0001, Table 3). This GCR rate increased another 26-fold upon deletion of MEC3 (P < 0.0001, Table 3). Screening of GCRs obtained from 66 independent cultures of the sgs1Δ mec3Δ exo1Δ mutant identified two CAN1/LYP1/ALP1 translocations, indicating a ~ 200-fold increase in the CAN1/LYP1/ALP1 translocation rate compared to the sgs1Δ mec3Δ mutant (3.5 × 10-6 versus 1.7 × 10-8).
Exo1 contains conserved N-terminal N- and I-nuclease domains, apparently separated by a short disordered linker, and binding sites for the mismatch repair (MMR) proteins Mlh1 and Msh2 have been located within the C-terminal half of Exo1 [72–74], which is predicted to be intrinsically disordered (Figure 1A). Four phosphorylation sites (S372, S567, S587, S692) required for the regulation of the DNA-damage response have also been located in the disordered C-terminus . To determine if the C-terminus of Exo1 plays a role in the suppression of genome instability in the sgs1Δ mutant we constructed a set of C-terminal deletions ranging from 100 to 400 residues (Figure 1A and 1B). We found that the C-terminal 260 residues of Exo1, making up 37% of the protein, play no major role in suppressing the accumulation of GCRs in the sgs1Δ mutant (Table 4). To test the possibility that the C-terminus with its binding sites for MMR proteins might be required for Exo1's role in mutation avoidance, but not for its role in suppressing GCRs, we utilized a fluctuation assay to determine the rate of accumulating canavanine resistance (Canr) mutations in strains expressing the various C-terminal Exo1 truncations (Table 5). As in the GCR assay, deletion of up to 260 residues had no effect on the Canr mutation rate (P = 0.3524) whereas deletion of 280 or more residues caused a null phenotype (P = 0.0001). Similarly, only deletion of 280 or more residues caused sensitivity to methyl methanesulfonate (MMS) (Figure 1C). No sensitivity to 200 mM hydroxyurea was observed for any of the exo1 mutants (Figure 1C). Thus, deletion of up to 260 residues caused a phenotype similar to wildtype in all assays tested here, whereas deletion of 280 or more residues caused a null (exo1Δ) phenotype.
In addition to providing MMR protein interaction sites, the C-terminus of Exo1 contains four phosphorylation sites (S372, S567, S587, S692), which were recently shown to be important for the regulation of the DNA damage checkpoint in response to uncapped telomeres in a cdc13-1 mutant . Unlike in a cdc13-1 mutant, we did not detect Exo1 phosphorylation in the sgs1Δ mutant (data not shown), and deletion of the C-terminal third of Exo1 (exo1-ΔC260), which contains three of the four phosphorylation sites (S567, S587, S692), had no effect on Exo1 function in the assays used here (Canr mutation rate, GCR assay, MMS sensitivity). The fourth phosphorylation site (S372) is present in both the exo1-ΔC260 mutant and the exo1-ΔC280 mutant and, therefore, is not responsible for the different phenotypes associated with the two alleles. Thus, the known phosphorylation sites in Exo1 do not appear to be required for Exo1's role in mutation avoidance, resistance to MMS or suppression of GCRs in a sgs1Δ mutant. Instead, it is likely that the ΔC280 deletion affects Exo1 nuclease activity directly by disrupting intramolecular interactions with the N-terminus. The loss of yet unknown posttranslational modifications in this segment of Exo1 or an indirect effect caused by the loss of interaction with other cellular factors could also lead to the deficiency of the exo1ΔC280 allele.
Besides the overall increase in genome instability, CAN1/LYP1/ALP1 rearrangements seen in the sgs1Δ mec3Δ mutant were also present in the sgs1Δ mec3Δ exo1Δ mutant. Normally, Exo1 and Sgs1 function in independent end resection pathways that cooperate in the processing of DSBs, especially the long-range resection of the 5'-strand [19, 20], and Marrero and Symington  recently showed that this extensive resection inhibits BIR in a plasmid-based assay. Besides upregulation of BIR, which was also accompanied by chromosome rearrangements, the exo1Δ sgs1Δ mutant was also more proficient in de novo telomere synthesis at HO-endonuclease-induced chromosome breaks [18, 21]. The combination of increased BIR and more efficient de novo telomere addition, both of which have been identified as major mechanisms for the healing of chromosome V breaks in the GCR assay [76, 77], likely also explains the remarkably strong accumulation of genome rearrangements originating from spontaneous DNA lesions in the exo1Δ sgs1Δ mutant studied here. Our study further adds that the exo1Δ sgs1Δ mutant has even greater potential for the accumulation of viable genome rearrangements, which is suppressed (~ 26-fold) in the sgs1Δ exo1Δ mutant by Mec3-dependent DNA-damage checkpoint functions (P < 0.0001). Nonhomologous endjoining does not appear to be a significant source for these genome rearrangements, as indicated by the lack of any effect of LIG4 gene deletion in mutants with various combinations of sgs1Δ, exo1Δ and mec3Δ mutations (e.g., GCR rate of sgs1Δ mec3Δ exo1Δ compared to sgs1Δ mec3Δ exo1Δ lig4Δ, P = 0.3953, Table 3); however, it is also plausible that in the absence of one repair pathway DNA lesions simply become substrates for various other available repair pathways.
Our results indicate that spontaneous, interchromosomal translocations between short regions of sequence identity (5-41 bp), such as those present in the CAN1, LYP1 and ALP1 genes used in our assay, are promoted by Mec1/Dun1/Rad59-dependent pathways whereas Tel1, Mec3 and Sgs1 act as independent suppressors (Figure 2). The requirement for Pol32 and Rad1 in the translocation process further suggests the need for extensive DNA synthesis, such as seen in BIR, and the removal of nonhomologous overhangs from annealed single-strands, critical for SSA and implicated in GCR formation. Exo1 nuclease is a suppressor of overall genome rearrangements as well as CAN1/LYP1/ALP1 translocations when cells lack Sgs1 or both Sgs1 and Mec3. That the disordered, C-terminal third is dispensable for Exo1 function in our assays further indicates that physical interaction with MMR proteins in this region and regulation of Exo1 function in response to DNA-damage are not important for Exo1's role in the suppression of spontaneous GCRs, mutation avoidance and resistance to MMS.
Yeast strains and media
All strains used in this study are derived from KHSY802 (MATa, ura3-52, trp1Δ63, his3Δ200, leu2Δ1, lys2Bgl, hom3-10, ade2Δ1, ade8, hxt13::URA3) or the isogenic strain of the opposite mating type. Desired gene deletions were introduced by HR-mediated integration of PCR products containing a selectable marker cassette flanked by 50-nt sequences complementary to the target locus . C-terminal truncations of Exo1 were constructed by replacing the desired DNA sequence at the chromosomal EXO1 locus with a myc-epitope encoding sequence amplified from pFA-13Myc.His3MX6 (a gift from Mark Longtine, Washington University, St. Louis). Expression of Exo1 truncation alleles was confirmed by Western blotting using monoclonal anti-c-myc antibody (Covance). All haploid strains with multiple gene deletions were obtained by sporulating diploids heterozygous for the desired mutations to minimize the risk of obtaining suppressors. This was especially important for combinations of mutations known to cause fitness defects, such as sgs1Δ and pol32Δ. Spore isolation was followed by genotyping of meiotic products by spotting on selective media or by PCR. All strains used in this study are listed in Table 6. Media for propagating yeast strains have been previously described [76, 77].
Sensitivity to DNA damaging agents HU and MMS
Cell cultures were grown in yeast extract/peptone/dextrose (YPD) media and adjusted to OD600 = 1. Tenfold dilutions were spotted on YPD, YPD supplemented with 0.05% methyl-methanesulfonate (MMS) and YPD supplemented with 200 mM hydroxyurea (HU). Colony growth was documented after incubation at 30°C for 3 days.
Rates of accumulating spontaneous gross-chromosomal rearrangements (GCRs) were determined by fluctuation analysis and the method of the median as previously described [77, 79]. Cells with GCRs were detected by their resistance to canavanine and 5-fluoro-orotic acid (Canr 5-FOAr) due to simultaneous inactivation of the CAN1 and URA3 genes, both located within a 12 kb nonessential region on the left arm of chromosome V. The median GCR rate is reported with 95% confidence intervals . GCR clones were screened by PCR to identify clones with rearrangements between CAN1 on chromosome V and LYP1 and/or ALP1 (collectively referred to as CAN1/LYP1/ALP1 rearrangements in the text), located in opposite orientations on the same arm of chromosome XIV . To determine the rate of accumulating spontaneous mutations that lead to inactivation of the CAN1 gene, 3-ml YPD cultures expressing wildtype Exo1 or C-terminal truncations of Exo1were grown overnight and aliquots were plated on synthetic media lacking arginine (US Biological) supplemented with 240 mg ml-1 canavanine (Sigma), and on YPD to obtain the viable cell count. Colonies were counted after two days of incubation at 30°C. At least twelve independent cultures from three isolates were analyzed per yeast strain. The median Canr mutation rate is reported with 95% confidence intervals . Statistical significance of differences in GCR rates was evaluated by using the Mann-Whitney test and programs from Dr. R. Lowry at Vassar College http://faculty.vassar.edu/lowry/VassarStats.html.
Protein extraction and Western blot analysis
Cells were grown in YPD until they reached OD600 = 0.5. Whole cell extract was prepared from 5 ml of culture using a standard trichloroacetic acid (TCA) extraction. Briefly, cells were pelleted, vortexed with glass beads for 10 minutes in 200 μl of 20% TCA, followed by centrifugation for 2 minutes. The pellet was resuspended in sample buffer and pH was neutralized with 2 M Tris buffer (pH 7.6). Proteins were separated by PAGE, transferred to a PVDF membrane and incubated with monoclonal anti-c-myc antibody (Covance) to detect myc-tagged proteins. Bands were visualized using ECL Plus Chemiluminescence kit (GE Healthcare).
5-fluoro-orotic acid resistant
Myung K, Chen C, Kolodner RD: Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae. Nature. 2001, 411: 1073-1076. 10.1038/35082608.
Myung K, Datta A, Chen C, Kolodner RD: SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination. Nat Genet. 2001, 27: 113-116. 10.1038/83673.
Myung K, Datta A, Kolodner RD: Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae. Cell. 2001, 104: 397-408. 10.1016/S0092-8674(01)00227-6.
Myung K, Kolodner RD: Suppression of genome instability by redundant S-phase checkpoint pathways in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 2002, 99: 4500-4507. 10.1073/pnas.062702199.
Myung K, Pennaneach V, Kats ES, Kolodner RD: Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability. Proc Natl Acad Sci USA. 2003, 100: 6640-6645. 10.1073/pnas.1232239100.
Putnam CD, Hayes TK, Kolodner RD: Specific pathways prevent duplication-mediated genome rearrangements. Nature. 2009, 460: 984-989. 10.1038/nature08217.
Putnam CD, Jaehnig EJ, Kolodner RD: Perspectives on the DNA damage and replication checkpoint responses in Saccharomyces cerevisiae. DNA Repair (Amst). 2009, 8: 974-982. 10.1016/j.dnarep.2009.04.021.
Putnam CD, Pennaneach V, Kolodner RD: Saccharomyces cerevisiae as a model system to define the chromosomal instability phenotype. Mol Cell Biol. 2005, 25: 7226-7238. 10.1128/MCB.25.16.7226-7238.2005.
Schmidt KH, Kolodner RD: Suppression of spontaneous genome rearrangements in yeast DNA helicase mutants. Proc Natl Acad Sci USA. 2006, 103: 18196-18201. 10.1073/pnas.0608566103.
Schmidt KH, Wu J, Kolodner RD: Control of Translocations between Highly Diverged Genes by Sgs1, the Saccharomyces cerevisiae Homolog of the Bloom's Syndrome Protein. Mol Cell Biol. 2006, 26: 5406-5420. 10.1128/MCB.00161-06.
Antoccia A, Kobayashi J, Tauchi H, Matsuura S, Komatsu K: Nijmegen breakage syndrome and functions of the responsible protein, NBS1. Genome Dyn. 2006, 1: 191-205.
Carney JP, Maser RS, Olivares H, Davis EM, Le Beau M, Yates JR, Hays L, Morgan WF, Petrini JH: The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell. 1998, 93: 477-486. 10.1016/S0092-8674(00)81175-7.
Varon R, Vissinga C, Platzer M, Cerosaletti KM, Chrzanowska KH, Saar K, Beckmann G, Seemanova E, Cooper PR, Nowak NJ, et al: Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell. 1998, 93: 467-476. 10.1016/S0092-8674(00)81174-5.
Ellis NA, Groden J, Ye TZ, Straughen J, Lennon DJ, Ciocci S, Proytcheva M, German J: The Bloom's syndrome gene product is homologous to RecQ helicases. Cell. 1995, 83: 655-666. 10.1016/0092-8674(95)90105-1.
Yu CE, Oshima J, Fu YH, Wijsman EM, Hisama F, Alisch R, Matthews S, Nakura J, Miki T, Ouais S, et al: Positional cloning of the Werner's syndrome gene. Science. 1996, 272: 258-262. 10.1126/science.272.5259.258.
Savitsky K, Bar-Shira A, Gilad S, Rotman G, Ziv Y, Vanagaite L, Tagle DA, Smith S, Uziel T, Sfez S, et al: A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science. 1995, 268: 1749-1753. 10.1126/science.7792600.
Morrow DM, Tagle DA, Shiloh Y, Collins FS, Hieter P: TEL1, an S. cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is functionally related to the yeast checkpoint gene MEC1. Cell. 1995, 82: 831-840. 10.1016/0092-8674(95)90480-8.
Lydeard JR, Lipkin-Moore Z, Jain S, Eapen VV, Haber JE: Sgs1 and exo1 redundantly inhibit break-induced replication and de novo telomere addition at broken chromosome ends. PLoS Genet. 2010, 6: e1000973-10.1371/journal.pgen.1000973.
Mimitou EP, Symington LS: Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature. 2008, 455: 770-774. 10.1038/nature07312.
Zhu Z, Chung WH, Shim EY, Lee SE, Ira G: Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell. 2008, 134: 981-994. 10.1016/j.cell.2008.08.037.
Marrero VA, Symington LS: Extensive DNA end processing by exo1 and sgs1 inhibits break-induced replication. PLoS Genet. 2010, 6: e1001007-10.1371/journal.pgen.1001007.
Cobb JA, Bjergbaek L, Gasser SM: RecQ helicases: at the heart of genetic stability. FEBS Lett. 2002, 529: 43-48. 10.1016/S0014-5793(02)03269-6.
Frei C, Gasser SM: The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci. Genes Dev. 2000, 14: 81-96.
Ira G, Malkova A, Liberi G, Foiani M, Haber JE: Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell. 2003, 115: 401-411. 10.1016/S0092-8674(03)00886-9.
Lee SK, Johnson RE, Yu SL, Prakash L, Prakash S: Requirement of yeast SGS1 and SRS2 genes for replication and transcription. Science. 1999, 286: 2339-2342. 10.1126/science.286.5448.2339.
Versini G, Comet I, Wu M, Hoopes L, Schwob E, Pasero P: The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication. Embo J. 2003, 22: 1939-1949. 10.1093/emboj/cdg180.
Bentley NJ, Holtzman DA, Flaggs G, Keegan KS, DeMaggio A, Ford JC, Hoekstra M, Carr AM: The Schizosaccharomyces pombe rad3 checkpoint gene. Embo J. 1996, 15: 6641-6651.
Carr AM: Control of cell cycle arrest by the Mec1sc/Rad3sp DNA structure checkpoint pathway. Curr Opin Genet Dev. 1997, 7: 93-98. 10.1016/S0959-437X(97)80115-3.
Cimprich KA, Shin TB, Keith CT, Schreiber SL: cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein. Proc Natl Acad Sci USA. 1996, 93: 2850-2855. 10.1073/pnas.93.7.2850.
Weinert TA, Kiser GL, Hartwell LH: Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev. 1994, 8: 652-665. 10.1101/gad.8.6.652.
Tercero JA, Diffley JF: Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature. 2001, 412: 553-557. 10.1038/35087607.
Merrill BJ, Holm C: A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants. Genetics. 1999, 153: 595-605.
Grushcow JM, Holzen TM, Park KJ, Weinert T, Lichten M, Bishop DK: Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required for normal meiotic recombination partner choice. Genetics. 1999, 153: 607-620.
Kato R, Ogawa H: An essential gene, ESR1, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae. Nucleic Acids Res. 1994, 22: 3104-3112. 10.1093/nar/22.15.3104.
Lydall D, Nikolsky Y, Bishop DK, Weinert T: A meiotic recombination checkpoint controlled by mitotic checkpoint genes. Nature. 1996, 383: 840-843. 10.1038/383840a0.
Greenwell PW, Kronmal SL, Porter SE, Gassenhuber J, Obermaier B, Petes TD: TEL1, a gene involved in controlling telomere length in S. cerevisiae, is homologous to the human ataxia telangiectasia gene. Cell. 1995, 82: 823-829. 10.1016/0092-8674(95)90479-4.
Ritchie KB, Mallory JC, Petes TD: Interactions of TLC1 (which encodes the RNA subunit of telomerase), TEL1, and MEC1 in regulating telomere length in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1999, 19: 6065-6075.
Sanchez Y, Desany BA, Jones WJ, Liu Q, Wang B, Elledge SJ: Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science. 1996, 271: 357-360. 10.1126/science.271.5247.357.
Melo JA, Cohen J, Toczyski DP: Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. Genes Dev. 2001, 15: 2809-2821.
Kondo T, Wakayama T, Naiki T, Matsumoto K, Sugimoto K: Recruitment of Mec1 and Ddc1 checkpoint proteins to double-strand breaks through distinct mechanisms. Science. 2001, 294: 867-870. 10.1126/science.1063827.
Makovets S, Blackburn EH: DNA damage signalling prevents deleterious telomere addition at DNA breaks. Nat Cell Biol. 2009, 11: 1383-1386. 10.1038/ncb1985.
Zhang W, Durocher D: De novo telomere formation is suppressed by the Mec1-dependent inhibition of Cdc13 accumulation at DNA breaks. Genes Dev. 24: 502-515.
Bashkirov VI, King JS, Bashkirova EV, Schmuckli-Maurer J, Heyer WD: DNA repair protein Rad55 is a terminal substrate of the DNA damage checkpoints. Mol Cell Biol. 2000, 20: 4393-4404. 10.1128/MCB.20.12.4393-4404.2000.
Flott S, Alabert C, Toh GW, Toth R, Sugawara N, Campbell DG, Haber JE, Pasero P, Rouse J: Phosphorylation of Slx4 by Mec1 and Tel1 regulates the single-strand annealing mode of DNA repair in budding yeast. Mol Cell Biol. 2007, 27: 6433-6445. 10.1128/MCB.00135-07.
Ehmsen KT, Heyer WD: Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease. Nucleic Acids Res. 2008, 36: 2182-2195. 10.1093/nar/gkm1152.
Mordes DA, Nam EA, Cortez D: Dpb11 activates the Mec1-Ddc2 complex. Proc Natl Acad Sci USA. 2008, 105: 18730-18734. 10.1073/pnas.0806621105.
Herzberg K, Bashkirov VI, Rolfsmeier M, Haghnazari E, McDonald WH, Anderson S, Bashkirova EV, Yates JR, Heyer WD: Phosphorylation of Rad55 on serines 2, 8, and 14 is required for efficient homologous recombination in the recovery of stalled replication forks. Mol Cell Biol. 2006, 26: 8396-8409. 10.1128/MCB.01317-06.
Zhou Z, Elledge SJ: DUN1 encodes a protein kinase that controls the DNA damage response in yeast. Cell. 1993, 75: 1119-1127. 10.1016/0092-8674(93)90321-G.
Huang M, Zhou Z, Elledge SJ: The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor. Cell. 1998, 94: 595-605. 10.1016/S0092-8674(00)81601-3.
Sanchez Y, Bachant J, Wang H, Hu F, Liu D, Tetzlaff M, Elledge SJ: Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science. 1999, 286: 1166-1171. 10.1126/science.286.5442.1166.
Gardner R, Putnam CW, Weinert T: RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast. Embo J. 1999, 18: 3173-3185. 10.1093/emboj/18.11.3173.
Chan JE, Kolodner RD: A genetic and structural study of genome rearrangements mediated by high copy repeat Ty1 elements. PLoS Genet. 7: e1002089-
Chen Q, Ijpma A, Greider CW: Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events. Mol Cell Biol. 2001, 21: 1819-1827. 10.1128/MCB.21.5.1819-1827.2001.
Teng SC, Chang J, McCowan B, Zakian VA: Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process. Mol Cell. 2000, 6: 947-952. 10.1016/S1097-2765(05)00094-8.
Teng SC, Zakian VA: Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae. Mol Cell Biol. 1999, 19: 8083-8093.
Malkova A, Ivanov EL, Haber JE: Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. Proc Natl Acad Sci USA. 1996, 93: 7131-7136. 10.1073/pnas.93.14.7131.
Signon L, Malkova A, Naylor ML, Klein H, Haber JE: Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break. Mol Cell Biol. 2001, 21: 2048-2056. 10.1128/MCB.21.6.2048-2056.2001.
Symington LS: Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev. 2002, 66: 630-670. 10.1128/MMBR.66.4.630-670.2002. table of contents
Sugawara N, Ira G, Haber JE: DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair. Mol Cell Biol. 2000, 20: 5300-5309. 10.1128/MCB.20.14.5300-5309.2000.
Schmidt KH, Viebranz E, Doerfler L, Lester C, Rubenstein A: Formation of complex and unstable chromosomal translocations in yeast. PLoS One. 2010, 5: e12007-10.1371/journal.pone.0012007.
Pannunzio NR, Manthey GM, Bailis AM: RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae. DNA Repair (Amst). 2008, 7: 788-800. 10.1016/j.dnarep.2008.02.003.
Onoda F, Seki M, Miyajima A, Enomoto T: Elevation of sister chromatid exchange in Saccharomyces cerevisiae sgs1 disruptants and the relevance of the disruptants as a system to evaluate mutations in Bloom's syndrome gene. Mutat Res. 2000, 459: 203-209.
Ooi SL, Shoemaker DD, Boeke JD: DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray. Nat Genet. 2003, 35: 277-286. 10.1038/ng1258.
Watt PM, Hickson ID, Borts RH, Louis EJ: SGS1, a homologue of the Bloom's and Werner's syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae. Genetics. 1996, 144: 935-945.
Louis EJ, Haber JE: The structure and evolution of subtelomeric Y' repeats in Saccharomyces cerevisiae. Genetics. 1992, 131: 559-574.
Lydeard JR, Jain S, Yamaguchi M, Haber JE: Break-induced replication and telomerase-independent telomere maintenance require Pol32. Nature. 2007, 448: 820-823. 10.1038/nature06047.
Fishman-Lobell J, Haber JE: Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1. Science. 1992, 258: 480-484. 10.1126/science.1411547.
Ivanov EL, Haber JE: RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1995, 15: 2245-2251.
Hwang JY, Smith S, Myung K: The Rad1-Rad10 complex promotes the production of gross chromosomal rearrangements from spontaneous DNA damage in Saccharomyces cerevisiae. Genetics. 2005, 169: 1927-1937. 10.1534/genetics.104.039768.
Bartsch S, Kang LE, Symington LS: RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates. Mol Cell Biol. 2000, 20: 1194-1205. 10.1128/MCB.20.4.1194-1205.2000.
Malagon F, Aguilera A: Yeast spt6-140 mutation, affecting chromatin and transcription, preferentially increases recombination in which Rad51p-mediated strand exchange is dispensable. Genetics. 2001, 158: 597-611.
Tran PT, Erdeniz N, Symington LS, Liskay RM: EXO1-A multi-tasking eukaryotic nuclease. DNA Repair (Amst). 2004, 3: 1549-1559. 10.1016/j.dnarep.2004.05.015.
Schmutte C, Sadoff MM, Shim KS, Acharya S, Fishel R: The interaction of DNA mismatch repair proteins with human exonuclease I. J Biol Chem. 2001, 276: 33011-33018. 10.1074/jbc.M102670200.
Tishkoff DX, Boerger AL, Bertrand P, Filosi N, Gaida GM, Kane MF, Kolodner RD: Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2. Proc Natl Acad Sci USA. 1997, 94: 7487-7492. 10.1073/pnas.94.14.7487.
Morin I, Ngo HP, Greenall A, Zubko MK, Morrice N, Lydall D: Checkpoint-dependent phosphorylation of Exo1 modulates the DNA damage response. Embo J. 2008, 27: 2400-2410. 10.1038/emboj.2008.171.
Chen C, Umezu K, Kolodner RD: Chromosomal rearrangements occur in S. cerevisiae rfa1 mutator mutants due to mutagenic lesions processed by double-strand-break repair. Mol Cell. 1998, 2: 9-22. 10.1016/S1097-2765(00)80109-4.
Schmidt KH, Pennaneach V, Putnam CD, Kolodner RD: Analysis of gross-chromosomal rearrangements in Saccharomyces cerevisiae. Methods Enzymol. 2006, 409: 462-476.
Gietz RD, Woods RA: Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol Biol. 2006, 313: 107-120.
Lea DE, Coulson CA: The distribution of the number of mutants in bacterial populations. J Genet. 1949, 49: 264-285. 10.1007/BF02986080.
Nair KR: Table of confidence intervals for the median in samples from any continuous population. Sankhya. 1940, 4: 551-558.
Mirzaei H, Syed S, Kennedy J, Schmidt KH: Sgs1 truncations inducegenome rearrangements but suppress detrimental effects of BLM overexpression in Saccharomyces cerevisiae. J Mol Biol. 2011, 405: 877-891. 10.1016/j.jmb.2010.11.035.
Schmidt KH, Viebranz EB, Harris LB, Mirzaei-Souderjani H, Syed S, Medicus R: Defects in DNA lesion bypass lead to spontaneous chromosomal rearrangements and increased cell death. Eukaryot Cell. 2010, 9: 315-324. 10.1128/EC.00260-09.
We thank the members of the Schmidt lab and Gary Daughdrill (University of South Florida) for helpful discussions, Richard Kolodner (Ludwig Institute for Cancer Research, University of California San Diego) for sending strains and Mark Longtine (Washington University, St. Louis) for plasmids. This work was supported by National Institutes of Health grant 5R01GM081425 to KHS.
The authors declare that they have no competing interests.
LD constructed yeast strains, performed experiments, analyzed data and performed statistical analyses. LH constructed yeast strains, performed experiments, analyzed data and performed statistical analyses. EV constructed yeast strains and performed experiments, KHS. designed the study, analyzed data and wrote the manuscript. All authors have read and approved the final manuscript.
Lillian Doerfler, Lorena Harris contributed equally to this work.
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Doerfler, L., Harris, L., Viebranz, E. et al. Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability. Genome Integrity 2, 8 (2011). https://doi.org/10.1186/2041-9414-2-8
- genome instability
- mitotic recombination
- DNA-damage checkpoint
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