Collision between a topoisomerase I-DNA intermediate and an advancing replication fork represents a unique form of replicative damage. We have shown previously that yeast H2A serine 129 is involved in the recovery from this type of damage. We now report that efficient repair also requires proteins involved in chromatid cohesion: Csm3; Tof1; Mrc1, and Dcc1. Epistasis analysis defined several pathways involving these proteins. Csm3 and Tof1 function in a same pathway and downstream of H2A. In addition, the pathway involving H2A/Csm3/Tof1 is distinct from the pathways involving the Ctf8/Ctf18/Dcc1 complex, the Rad9 pathway, and another involving Mrc1. Our genetic studies suggest a role for H2A serine 129 in the establishment of specialized cohesion structure necessary for the normal repair of topoisomerase I-induced DNA damage.
DNA topoisomerase I (hereafter Top1) acts to prevent the buildup of superhelical strain around the elongating replication fork by transiently cleaving and religating one strand of the duplex DNA via a covalent 3′ phosphotyrosyl enzyme-DNA intermediate (Champoux 2001). In the presence of the antineoplastic drug and reversible inhibitor camptothecin (hereafter CPT), the half-life of the Top1–DNA cleavage complexes, which are normally short-lived catalytic intermediates, is increased due to a slowed rate of DNA religation (Svejstrup et al. 1991). Cytotoxic lesions are thought to occur when the advancing replication machinery encounters a drug-stabilized enzyme–DNA complex (Hsiang et al. 1989) Such collisions generate a unique form of replicative damage that produces DNA double-strand breaks (hereafter DSBs) trapping Top1 on the DNA in irreversible suicide complexes (Pommier et al. 2003).
In mammals, one of the earliest steps detectable after formation of DSBs is the rapid phosphorylation of histone H2AX in the chromatin adjacent to the break site (Rogakou et al. 1999). The phosphorylated H2AX (hereafter γ-H2AX) forms foci (Paull et al. 2000; Celeste et al. 2002). The phosphorylated serine is in an SQE consensus target motif recognized by three PI-3 kinases involved in DNA DSB repair, DNA-PK, ATM, and ATR (Shiloh 2001). While H2AX is a minor histone species in mammals, mice lacking H2AX suffer from unstable genomes, and primary MEF and T-cell cultures from these mice contain cells with multiple chromosome abnormalities (Celeste et al. 2002). Recently, H2AX phosphorylation was shown to promote efficient repair of a chromosomal DSB by sister-chromatid-templated recombination (Xie et al. 2004). The yeast histone species homologous to H2AX, H2A1, and H2A2, comprise ∼95% of the total yeast H2A complement. Multiple H2A phosphorylation sites on serine and threonin residues have been characterized in budding yeast (S122, S129, and T126) (Wyatt et al. 2004; Harvey et al. 2005). These modifications have roles in various cellular mechanisms such as DSB repair or telomere position effects (Wyatt et al. 2004; Harvey et al. 2005). Studies also show functional redundancy of these three phosphorylation sites, illustrating the complexity of the role of H2A in cellular processes (Wyatt et al. 2004). Yeast H2A1 and H2A2 (hereafter H2A) are phosphorylated on serine 129 in response to DNA damage, including Top1-induced DNA damage (Downs et al. 2000; Redon et al. 2003). H2A Ser 129 is an essential component for the efficient repair of DNA DSBs induced during replication by camptothecin (CPT). Yeast strains lacking H2A serine 129 (h2a1-s129a and h2a2-s129a, hereafter H2A-S129), are hypersensitive to CPT. Moreover, the pathway involving H2A-S129 for such lesions is epistatic with RAD52 but independent of the RAD9/RAD24 checkpoint (Redon et al. 2003).
Csm3, Mrc1, and Tof1 were originally identified as checkpoint proteins involved in transmitting the DNA replication arrest to downstream effectors (Alcasabas et al. 2001; Foss 2001; Tanaka and Russell 2001; Tong et al. 2004), by activating the Rad53 kinase in response to MMS. Genetic analysis showed that Tof1 and Rad9 have overlapping functions in response to MMS- and UV-induced DNA damage (Foss 2001). Tof1 and Csm3 interact in a two-hybrid assay and by coimmunoprecipitation (Ito et al. 2001; Mayer et al. 2004) and csm3Δ mutants have mitotic phenotypes similar to that of tof1Δ mutants regarding activation of Rad53 and cell-cycle arrest following MMS treatment (Tong et al. 2004). Other studies showed that the Saccharomyces pombe Swi1–Swi3 complex (Tof1–Csm3 homologs) is required for survival after fork arrest (Noguchi et al. 2003; Noguchi et al. 2004). Tof1 travels with the replication fork and is needed to restrain fork progression when DNA synthesis is inhibited by hydroxyurea (HU) (Katou et al. 2003; Osborn and Elledge 2003). This function is shared with Mrc1 (Katou et al. 2003). Tof1 belongs to a large protein family that was first defined by metazoan Tim1 (Timeless) (Chan et al. 2003). Drosophila melanogaster and mammalian Tim1s are implicated in circadian rhythmic oscillations (Barnes et al. 2003), whereas the Caenorhabditis elegans Tim1 is required for proper chromosome cohesion and segregation. Recent studies have uncovered a partial sister- chromatid cohesion defect in tof1Δ, csm3Δ, and mrc1Δ cells (Mayer et al. 2004; Xu et al. 2004). This phenotype is interesting in light of the role of C. elegans Tim1 (Tof1/Swi1 homolog) in chromosome cohesion and segregation (Chan et al. 2003). A mouse Csm3/Swi3 homolog TIPIN was identified as interacting with Tim1 (Gotter 2003). Recent studies show that, in budding yeast, DSB induction elicits recruitment of cohesin to the chromatin 50–100 kb around the lesion (Ström et al. 2004; Ünal et al. 2004) and that histone H2A serine 129 phosphorylation is required for cohesin binding (Ünal et al. 2004).
We decided to investigate a possible functional and structural relationship between H2A serine 129 and the Csm3–Tof1 complex. Here we report that these three factors work in the same pathway for repair of Top1-induced DNA damage in the presence of Mrc1 but independently in its absence. The pathways defined by H2A-S129A and Csm3–Tof1 are independent from Rad9 and Mrc1 pathways. Finally, we show that the H2A-S129 pathway is redundant with the pathway including the Ctf8/Ctf18/Dcc1 complex, a complex also involved in chromatid cohesion. We propose that H2A-S129 could initiate chromatid cohesion specifically involved in the repair of Top1-induced DNA damage.
MATERIALS AND METHODS
Yeast methods and strains:
All methods for the manipulation of yeast were performed according to standard methods. The strains used in this study are derived from W303 and are listed in Table 1.
Sensitivity assays on plates:
Stock solutions of CPT (Sigma, St. Louis) were prepared at a concentration of 6 mg/ml in dimethylsulphoxyde (DMSO). Stock solutions of hydroxyurea (Sigma) were prepared at 2 m in H2O. Aliquots of the stocks were spread on YPD plates to give the final concentrations desired; control plates received the same volume of DMSO for CPT or H2O for HU. Dilutions (10-fold) from overnight cultures were spotted on the plates and grown at 30° for 2–3 days.
Cells were collected by centrifugation, placed in ice, and exposed to the indicated amount of ionizing radiation from a 137Cs source (Mark I irradiator; J. L. Shepherd and Associates) at a rate of 15.7 Gy/min.
Protein extraction and immunoblotting:
Cell extracts were prepared as previously described (Redon et al. 2003). For H2A Western blot analysis, proteins were separated using 13% SDS–polyacrylamide gels and blotted onto polyvinylidene difluoride membranes. Membranes were incubated with an antibody raised against a conjugated peptide composed of the 10 carboxy-terminal amino acids of H2A that contain the phosphorylated serine located four residues from the carboxyl terminus [AKATKAS(P)QEL]. Procedures for Rad53 analysis have been described previously (de la Torre-Ruiz et al. 1998).
Paraformaldehyde was added to 2–5 ml cultures to a final concentration of 4% and incubated at room temperature for 1 hr with gentle agitation. Cells were washed five times with a solution containing 5 mm MgCl2, 40 mm KH2PO4 and resuspended in 0.5 ml of digestion buffer containing 5 mm MgCl2, 40 mm KH2PO4, and 1.2 m sorbitol. Zymolase (1 mg/ml final concentration) was added and cells were incubated at 30° for 45–60 min. After a wash with digestion buffer, cells were spotted on slides precoated with poly-l-lysine (Sigma) and incubated 15–30 min. Slides were then incubated in blocking buffer (1.5% BSA in PBS, 0.5% Tween 20, and 0.1% Triton X-100) for 30 min. Incubation with the primary antibody directed against H2A phosphoserine 129 (1:1000 dilution) was performed overnight at room temperature. The slides were then washed two to three times with PBS, once with blocking buffer and then incubated for 1 hr at room temperature with a secondary antibody conjugated with the green fluorescent dye Alexa 488 (Molecular probes, 1:500 dilution in blocking buffer). After three washes with PBS, slides were incubated for 1 hr at 37° in a solution containing 0.5 mg/ml RNAse A and 200 μg/ml PI to detect DNA. Slides were then mounted with antifade medium. Samples were visualized using a PCM2000 laser-scanning confocal microscope (Nikon, Garden, City, NY).
Assessing sister-chromatid cohesion:
Lac repressor–GFP/Lac operator repeat strains were grown exponentially in YPD before arresting in G2/M with 15 μg/ml nocodazole (Sigma). Cells were fixed with an equal volume of fresh 4% paraformaldehyde for 5 min at room temperature, washed once with PBS, and resuspended in PBS for cohesion assessment by fluorescence microscopy.
H2A serine 129 is in the same epistasis group with Tof1 and Csm3 relative to resistance to Top1-induced DNA damage:
To simplify, hta-S129A refers to the hta1-S129A hta2-S129A double mutant. During a screen aimed at identifying mutants that are particularly dependent on RAD9 for surviving DNA damage, Foss (2001) recovered a TOF1 mutant. Deletion of both TOF1 and RAD9 genes conferred synergistic sensitivity to MMS, UV, and HU (Foss 2001), similar to the relationship we reported for H2A-S129 and RAD9 (Redon et al. 2003). To explore a potential link between H2A-S129 and TOF1, we challenged hta-S129A and tof1Δ mutant strains with a variety of genotoxic agents (Figure 1A). Since Tof1 is known to interact with Csm3 (Ito et al. 2001; Mayer et al. 2004), we also examined csm3Δ mutant strains.
Several DNA-damaging agents are known to create distinct lesions that directly or via repair can induce DNA DSBs. Gamma IR directly generates single-strand breaks and DSBs via a free radical mechanism. In the case of UV light, the induced cyclobutane pyrimidine dimers and 6-4 photoproducts undergo excision repair that may induce DSBs (Friedberg et al. 1995). HU leads to nucleotide pool depletion, inhibiting DNA synthesis, and inducing DSBs (Merrill and Holm 1999).
While hta-S129A, csm3Δ, and tof1Δ cells are weakly sensitive to IR (500 Gy ∼35–40 DSBs), UV radiation, or HU (Figure 1A), they are similarly hypersensitive to CPT, suggesting that these three proteins may function together to process Top1–DNA complexes at stalled replication forks. This possibility was examined with double- and triple-mutant strains (Figure 1B). We found that the sensitivities of csm3Δ hta-S129A, csm3Δ tof1Δ, and tof1Δ hta-S129A double mutants to CPT are the same as the csm3Δ single mutant. In addition, the sensitivity of the tof1Δ csm3Δ hta-S129A triple mutant to CPT was the same as the csm3Δ single mutant, indicating that Csm3, Tof1, and H2A-S129 function in the same pathway for the repair of CPT-induced DNA damage.
Csm3 and Tof1 function downstream of H2A serine 129:
As CSM3, TOF1, and H2A-S129 show epistasis toward CPT, we hypothesized that Csm3 and/or Tof1 could be involved in H2A serine 129 phosphorylation in response to Top1-induced DNA damage. We examined strains bearing deletions for CSM3 and TOF1 for increased H2A serine 129 phosphorylation in the presence or absence of HU, CPT, or IR treatment (Figure 2A). In the presence of CPT or after treatment with γ-radiation, an increased signal was detected in wild-type, csm3Δ, and tof1Δ cells. We also observed a weak signal for H2A serine 129 phosphorylation in untreated cells, presumably due to DNA lesions produced during the replication process itself (Tishkoff et al. 1997; Zou and Rothstein 1997; Symington 1998; Chen et al. 1999; Lisby et al. 2001). We next sought to examine whether deletion of CSM3 or TOF1 could perturb the distribution of phosphorylated H2A in the nucleus after DNA damage by CPT. Following DNA damage in mammalian cells, H2AX-phosphorylated molecules appear in discrete nuclear foci within minutes after exposure to exogenous DNA damage agents (Rogakou et al. 1999). Other observations concluded that each focus represents one DNA DSB (Sedelnikova et al. 2002). For microscopic observations, asynchronous yeast cultures of wild-type (hereafter wt), hta-S129A, csm3Δ, and tof1Δ mutants were grown with or without 20 μm CPT for 60 min and then processed for immunocytochemistry. The results showed that phosphorylated H2A localizes into foci throughout the nucleus (Figure 2B). Cells carrying the hta-S129A mutation did not show any foci, even after treatment with CPT. In contrast, CPT treatment resulted in the formation of increasing numbers of discrete phosphorylated H2A foci throughout the nuclei of wt, csm3, and tof1Δ cells. These results show that the DNA damage-dependent phosphorylation of H2A serine 129 was not lost in strains with mutations in CSM3-TOF1 epistasis group. However, some csm3Δ and tof1Δ cells show abnormally high- phosphorylated H2A labeling. In fact, in the nuclei of csm3Δ and tof1Δ cells, between 10 and 20% nuclei showed full staining with antiphosphorylated H2A (see supplemental Figure 1 at http://www.genetics.org/supplemental/). These observations can be compared to Western blotting experiments where a higher signal for H2A serine 129 phosphorylation was observed for csm3Δ and tof1Δ cells (Figure 2A). These data suggest that some cells carrying CSM3 or TOF1 deletions suffered high intranuclear dysfunctions. The lack of CSM3 or TOF1 could cause increased DNA damage that, in turn, causes increased H2A serine 129 phosphorylation. Nonrepairable DNA lesions in these cells could lead to a cellular mechanism such as apoptosis (Wissing et al. 2004) and thus DNA fragmentation and H2A serine 129 phosphorylation.
The H2A-S129/Csm3/Tof1 epistasis group is independent of the Rad9/Rad24 group:
Previous genetic studies indicated that tof1Δ and rad9Δ conferred synergistic sensitivity to MMS, UV, and HU, and that TOF1's contribution to DNA damage response was restricted to S phase. Moreover, strains lacking both TOF1 and RAD17, RAD24, or MEC3 were also HU sensitive, even though none of these single mutants appear to be (Foss 2001). We reported similar observations when we examined the role of H2A-S129 in response to S-phase-induced DNA damage (Redon et al. 2003).
The increased CPT-induced killing of rad24Δ hta-S129A and rad9Δ hta-S129A double mutant strains relative to the single mutants suggested that H2A-S129 functions independent of the RAD9–RAD24 checkpoint pathway and prompted us to examine whether the CSM3–TOF1 epistasis group, along with H2A-S129, is involved in a pathway independent of the RAD9–RAD24 pathway. If CPT concentrations are decreased to a point at which the rad9Δ, hta-S129A, csm3Δ, csm3Δ hta-S129A, csm3Δ tof1Δ, and csm3Δ tof1Δ hta-S129A mutants show no sensitivity over wt, the addition of rad9Δ with any of the other mutants leads to the same greatly enhanced CPT sensitivity (Figure 3). These observations provide evidence that H2A-S129, CSM3, and TOF1 work together in a pathway independent from the RAD9–RAD24 pathway for Top1-induced DNA damage repair. The same results were found using another methodology for quantitative analysis of CPT sensitivity (see supplemental Figure 2 at http://www.genetics.org/supplemental/), which determine the sensitivity to different amounts of drug on the basis of exposure to different amounts of drug for a period of 10 doubling times (Vance and Wilson 2002).
The H2A-S129/Csm3/Tof1 and Rad9/Rad24 epistasis groups are independent of Mrc1:
Because Tof1 and Mrc1 are part of the replication fork machinery and involved in chromatid cohesion, we next examined the relationship between Mrc1 and the [H2A-S129/Csm3/Tof1] and Rad9 pathways for the repair of Top1-mediated DNA damage. In contrast to tof1Δ cells, mrc1Δ mutant strains do not exhibit sensitivity to CPT (Figures 1A and 4A). Additionally, the deletion of MRC1 alleviates the CPT sensitivity of hta-S129A strains (Figure 4A) but increases the sensitivity of csm3Δ and tof1Δ strains (Figure 4B). Furthermore, the deletion of MRC1 exacerbates the CPT sensitivity of hta-S129A strains when CSM3 and TOF1 are absent: csm3Δ mrc1Δ tof1Δ hta-S129A is much more sensitive than hta-S129A (Figure 4C). This observation suggested that Csm3 and Tof1 play a role in the rescue of hta-S129A cells when MRC1 is deleted. Moreover, the addition of an MRC1 deletion in rad9Δ cells leads to a further increase in sensitivity to CPT compared to either single mutant (Fig.4D). These results show that MRC1 plays a role in CPT resistance when CSM3/TOF1, H2A, or RAD9 are altered. While we previously showed epistasis between H2A-S129 and Csm3–Tof1, these observations further suggest that Csm3 and Tof1 define a supplementary pathway when MRC1 is absent.
To help understand the relationship between H2A-S129, MRC1, and RAD9, we next examined the sensitivity of rad9Δ, rad9Δ hta-S129A, mrc1Δ rad9Δ, mrc1Δ rad9Δ sml1Δ, and mrc1Δ rad9Δ sml1Δ hta-S129A mutants (Figure 4D) but could not determine the sensitivity of mrc1Δ rad9Δ hta-S129A strains, as cells carrying this genotype proved to be inviable (Figure 5). Viability was restored by the deletion of SML1 (see supplemental Figure 3 at http://www.genetics.org/supplemental/). We noted that, despite the marked sensitivity of the mrc1Δ rad9Δ strain to low doses of CPT, these cells were significantly more sensitive when carrying additional H2A and SML1 mutations (Figure 4D). The increased sensitivity observed in the mrc1Δ rad9Δ sml1Δ hta-S129A strain compared to the mrc1Δ rad9Δ strain is not due to alteration of SML1, as mrc1Δ rad9Δ and mrc1Δ rad9Δ sml1Δ strains showed the same sensitivity to the drug (Figure 4D). These observations showed a synergistic effect of H2A-S129, MRC1, and RAD9 toward CPT and suggested that H2A-S129, Mrc1, and Rad9 function in three redundant pathways for the repair of DSBs generated by Top1. The relationship between H2A-S129, Csm3, Mrc1, Tof1, and Rad9 will be described below.
The Csm3–Tof1 complex and H2A-S129 possess distinct biochemical functions for cellular growth:
Mrc1 has been reported to have a synthetic-lethal interaction with Rad9 (Alcasabas et al. 2001; Tong et al. 2004). In our hands, the mrc1Δ rad9Δ mutant is viable (Figure 5). This difference with previous work is probably due to differences in strain genetic backgrounds. In contrast, we could not obtain an mrc1Δ rad9Δ hta-S129A triple mutant (Figure 5). We observed the same synthetic-lethal interaction with CSM3 and TOF1, as csm3Δ mrc1Δ rad9Δ and the mrc1Δ rad9Δ tof1Δ triple mutants could not be obtained (Figure 5). To our knowledge, it is the first time that cells carrying the hta-S129A mutation are involved in a synthetic lethality interaction. As described above, the mrc1Δ rad9Δ hta-S129A lethality was suppressed by introducing a SML1 deletion. In S. cerevisiae, sml1 affects various cellular processes in a manner analogous to overproducing the large subunit ribonucleotide reductase RNR1. In fact, Sml1 inhibits dNTP synthesis post-translationally by binding directly to Rnr1, and sml1Δ mutants show increased levels of dNTP pools compared to wild type (Zhao et al. 1998). We observed that, unlike the mrc1Δ rad9Δ hta-S129A triple mutant, an SML1 deletion does not suppress the lethality of csm3Δ mrc1Δ rad9Δ or mrc1Δ rad9Δ tof1Δ triple mutants under standard growth conditions (see supplemental Figure 3 at http://www.genetics.org/supplemental/). Although H2A-S129 and Csm3–Tof1 show epistasis in resistance to CPT, these observations suggest that H2A-S129 and Csm3–Tof1 possess distinct biochemical functions for cellular growth, functions also not shared by either MRC1 or RAD9. These observations also suggest that Mrc1 possesses a function for cellular growth not shared by H2A-S129, because rad9Δ tof1Δ hta-S129A is alive, rad9Δ tof1Δ mrc1Δ is dead, rad9Δ csm3Δ hta-S129A is alive, and rad9Δ csm3Δ mrc1Δ is dead (Figure 5).
Unlike Csm3, H2A-S129 is not required for efficient sister-chromatid cohesion:
Because we observed that H2A-S129 and Csm3–Tof1 have functional similarities for the repair of Top1-induced replicative DNA damage, and that CSM3 and TOF1 deletion mutants have sister-chromatid cohesion defects (Mayer et al. 2004; Xu et al. 2004), we examined whether the DNA repair deficiency we observed in hta-S129A cells was due to a sister-chromatid cohesion defect. We first analyzed sister-chromatid cohesion directly in hta-S129A, mrc1Δ, and csm3Δ strains. Each of these strains was modified to carry both a Lac repressor–GFP fusion and a Lac operator repeat integrated in the LEU2 locus (∼20 kb away from the centromere of chromosome III). Cells were grown logarithmically, arrested in G2/M, fixed, and then scored to determine the number of GFP dots in each cell. The csm3Δ and mrc1Δ mutants showed more cells with two dots than did wt or hta-S129A. (Figure 6A). Cells with chromatid cohesion defects, such as Ctf8–RFC complex mutants, are also sensitive to benomyl (Mayer et al. 2001). As Dcc1 is a component of the Ctf8–RFC complex, we used a dcc1Δ strain, along with csm3Δ, mrc1Δ, and tof1Δ strains as positive controls. Our assay showed that csm3Δ, mrc1Δ, tof1Δ and, dcc1Δ cells, but not hta-S129A cells, were sensitive to benomyl (Figure 6B). These observations rule out a preexisting chromatid cohesion defect in hta-S129A cells, and therefore we can assume that the CPT sensitivity observed in hta-S129A cells is not primarily due to a chromatid cohesion defect. In contrast, we found that mrc1Δ cells are not sensitive to CPT (Figure 1A), but have a sister-chromatid cohesion defect. This observation shows that a sister-chromatid defect is not enough to lead to CPT sensitivity.
H2A-S129 and DCC1 function in redundant pathways for the repair of DSBs generated by Top1:
Csm3 and Tof1 have been identified as synthetic-lethal partners of Ctf8 (Mayer et al. 2004), a component of Ctf18–RFC, an alternative RFC complex composed of Rfc2, Rfc3, Rfc4, Rfc5, Ctf8, Ctf18, and Dcc1. One function of Ctf18-RFC is to initiate switching to DNA polymerase-σ during replication (Mayer et al. 2004). Moreover, like the Csm3–Tof1 complex, Ctf8–RFC is required for proper sister-chromatid cohesion (Hanna et al. 2001; Mayer et al. 2001; Naiki et al. 2001). As Csm3 and Tof1 are lethal with Ctf8, a partner of Dcc1, we hypothesized that an alteration of H2A-S129, in combination with DCC1 deletion, would show synthetic interaction and/or would confer additional sensitivity to CPT. First, we compared the CPT sensitivities of hta-S129A, csm3Δ, csm3Δ hta-S129A, dcc1Δ, and dcc1Δ hta-S129A strains (Figure 7A). The sensitivity of hta-S129A and dcc1Δ at 2 μm CPT was comparable to the wild type. In contrast, dcc1Δ hta-S129A mutant cells exhibited a marked increase in sensitivity to the drug (>100-fold), confirming that H2A and Dcc1 function in alternative pathways for the repair of CPT-induced DNA damage. As previously described in this study, such a synergistic effect was not observed between hta-S129A and csm3Δ (Figure 7A). Moreover, while synthetic-lethal interactions were observed between DCC1 and [CSM3, MRC1, TOF1], the dcc1Δ hta-S129A double mutant is viable and does not show a synthetic growth defect compared to either single mutant (see supplemental Figure 4 at http://www.genetics.org/supplemental/). As previously observed, these results let us consider that H2A-S129 and Csm3–Tof1 have functions, distinct from that necessary for repair of Top1-induced DNA damage. As we observed that both H2A-S129/Dcc1 and H2AS129/Rad9 are redundant for CPT-induced DNA damage, we next examined whether Dcc1 and Rad9 could be in a same pathway for the repair of this type of DNA damage. Despite the marked sensitivity of the rad9Δ hta-S129A and dcc1Δ hta-S129A mutants, these cells were significantly less sensitive than the dcc1Δ rad9Δ hta-S129A triple mutant (Figure 7B). These observations suggest that Dcc1, Rad9, and H2A-S129 are situated in three different pathways for the repair of DSBs generated by the action of Top1. We note that, even if Dcc1 and Rad9 are in different pathways, the double-mutant dcc1Δ rad9Δ is not more sensitive that the single dcc1Δ mutant. This observation can be explained by the fact that Rad9 (acting in G2/M) is not needed in Top1-induced DNA damage repair when the S-phase checkpoint-blind DNA damage repair machinery, including H2A-S129, is not compromised (Redon et al. 2003).
Because H2A-S129 and RAD9, on one hand, and TOF1 and RAD9, on the other hand, function as redundant pathways for repairing Top1-replicative damage and hydroxyurea-induced DNA damage (Foss 2001; Redon et al. 2003), we hypothesized that H2A-S129 and TOF1 could be a part of a similar pathway for DNA damage repair. Extensive genetic analysis revealed that, H2A-S129, Tof1, and Csm3, a protein that physically interacts with Tof1, function in the same pathway for the repair of CPT-induced DNA damage. The pathway involving [H2A-S129-TOF1-CSM3] is redundant with the pathways involving MRC1 and RAD9. However, our results let us consider that the Csm3–Tof1 protein complex is also involved in a mechanism independent from H2A-S129 when MRC1 is altered (Figure 8). In our model, Mrc1 could negatively regulate Csm3–Tof1. In fact, it is known that mrc1 or tof1 mutants show altered replication machinery (Katou et al. 2003; Zegerman and Diffley 2003). Thus, the replication fork rearrangement produced by mrc1 deletion could provide a new role for the Tof1-Csm3 complex in response to CPT. Moreover, the fact that H2A-S129 and CSM3/TOF1 can be in separate pathways for the repair of Top1-replicative damage when MRC1 is altered is supported by genetic interaction studies showing that H2A-S129 and CSM3/TOF1 can be in redundant pathways for normal cell growth.
Top1-induced DNA damage is repaired in a checkpoint-silent way in budding yeast, as no Rad53 hyperphosphorylation and no S-phase arrest are observed (Redon et al. 2003). In fact, repair of these lesions is already occurring during S-phase progression and cells undergo only a short arrest in G2/M. Genes involved in the G2/M checkpoint, such as RAD9, are crucial for the repair of Top1-replicative damage when H2A serine 129 phosphorylation was abolished (Redon et al. 2003). We show in this study that the same function can be ascribed to Csm3, Tof1, Mrc1, and Rad9, as csm3Δ rad9Δ, rad9Δ tof1Δ, and mrc1Δ rad9Δ double mutants show synergistic sensitivity when compared to single mutants.
A recent study showed that the localization of repair proteins to breaks is not likely to be the main function of H2A serine 129 phosphorylation (Shroff et al. 2004). The authors showed that H2A phosphorylation forms in a large (50 kb) region surrounding the DNA DSB site. Remarkably, very little H2A phosphorylation is present within 1–2 kb of the break. In contrast, this region contains almost all the Mre11 and other repair proteins that bind as a result of the break. In parallel, other works showed that the DNA damage response pathway is required for the formation of a large cohesin domain surrounding a DSB site (Ström et al. 2004; Ünal et al. 2004). Cohesin recruitment around the DSB requires H2A serine 129 phosphorylation by Mec1 and Tel1 (Ünal et al. 2004). Interestingly, we previously showed that H2A-S129 is epistatic to Mec1 and Tel1 for the repair of Top1-induced DNA damage (Redon et al. 2003).
DSB-specific cohesin loading is required for the efficient postreplicative repair of DSBs (Ström et al. 2004). Postreplicative repair requires DSB-induced cohesin binding to promote local sister-chromatid cohesion (Ünal et al. 2004). In S phase, the establishment of cohesion requires the close proximity of sister chromatids, which is achieved immediately behind the replication fork. It is interesting to note that Tof1 and Mrc1, both involved in the repair of Top1-induced DNA damage and in chromatid cohesion, are a structural part of the replication machinery (Katou et al. 2003). Also, the C. elegans Tim1, a Tof1 homolog, is moving with the replication fork and required for proper chromosome cohesion (Chan et al. 2003). Csm3, also involved in sister-chromatid cohesion, interacts physically with Tof1 and could be part of the replicative machinery. As the Tof1–Csm3 complex is involved in sister-chromatid cohesion on one hand, and H2A-S129 is recruiting cohesin proteins at the DSB sites on the other hand, we propose that the role of H2A-S129 is to allow the formation of a specialized repair cohesion structure, whose formation involves Csm3–Tof1. This mechanism would work in parallel with other(s) cohesion mechanisms involving Mrc1 and/or the Ctf18/Dcc1 complex. For these reasons, further studies will be needed to determine if recruitment of cohesins such as Mcd1 around the DSB requires the presence of Csm3 and/or Tof1.
As it appears that the protein complexes related to Tof1–Csm3 are broadly conserved among eukaryotes, it will be interesting to determine whether these complexes are involved, together with H2AX phosphorylation, in the repair of DNA replication-induced DNA damage in mammalian cells.
↵1 Present address: IPBS, CNRS UMR 5089, 205 Route de Narbonne, 31077 Toulouse Cedex, France.
Communicating editor: M. Lichten
- Received May 26, 2005.
- Accepted October 2, 2005.
- Copyright © 2006 by the Genetics Society of America