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Constance Alabert,2
Geraldine W. Toh,1
Rachel Toth,1
Neal Sugawara,3
David G. Campbell,1
James E. Haber,3
Philippe Pasero,2 and
John Rouse1*
MRC Protein Phosphorylation Unit, James Black Centre, University of Dundee, Dundee DD1 5EH, United Kingdom,1 Institute of Human Genetics, CNRS UPR 1142, 141 Rue de la Cardonille, 34396 Montpellier, France,2 Rosenstiel Basic Medical Sciences Research Centre, Waltham, Massachusetts3
Received 22 January 2007/ Returned for modification 1 June 2007/ Accepted 28 June 2007
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ABSTRACT |
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INTRODUCTION |
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Replisomes blocked by DNA damage can bypass lesions by at least two different mechanisms: translesion synthesis (TLS) across the damaged base by TLS polymerases and error-free bypass, also known as "template switching" (34, 47). Template switching is thought to involve unpairing of the nascent strands from the parental template and their subsequent annealing (20, 39). The nascent strand that had been blocked is then elongated using the nascent sister strand as template. The resulting structures may be converted to pseudo-dHJs, in a Rad51/Rad52-dependent manner, and cells lacking Sgs1 accumulate these pseudo-dHJs presumably because it normally helps to dissolve them (27). There is experimental evidence to support template switching at blocked replisomes in yeast (53), but the mechanisms involved are unclear, as are many of the relevant genes.
Despite these important roles, Sgs1 is not essential for cell viability. However, a screen for genes required for viability in the absence of SGS1 (or TOP3) identified six "SLX" genes. The products of these genes form three heterodimeric complexes in cells: Slx2 (Mms4)/Slx3 (Mus81), Slx5/Slx8, and Slx1/Slx4 (31). The precise cellular roles of Slx5 and Slx8 are unclear, although cells lacking either protein showed an increased rate of gross chromosomal rearrangements (52). Mms4-Mus81 is a structure-specific endonuclease that, at least in vitro, preferentially cleaves branched DNA structures resembling structures that arise during recombinational processing of stalled replication forks (2, 23). The synthetic lethality of sgs1 mus81 cells is suppressed when RAD52 is deleted (2, 10), suggesting Mus81 cleaves dHJs when they are not dissolved by Sgs1 during recombinational processing of stalled replisomes.
Slx1-Slx4 from budding yeast and fission yeast is also a structure-specific endonuclease with preference in vitro for branched DNA substrates, especially simple-Y, 5'-flap, or replication fork-like structures (7, 14). Slx1-Slx4 is likely to define a pathway distinct from Mms4-Mus81, because the synthetic lethality of sgs1
slx1 or sgs1 slx4 cells cannot be rescued by deletion of RAD52 (2, 10). Slx4 has no obvious catalytic or structural motifs, apart from a cryptic SAP domain, but Slx1 has a PHD-type zinc finger and is the founding member of a conserved family of nucleases defined by a UvrC-intron-endonuclease (URI) domain (14).
While some of the cellular functions of Slx1 and Slx4 proteins are likely to overlap, given that these proteins interact and are both required for viability in the absence of Sgs1, cells lacking Slx4 are hypersensitive to DNA alkylation damage whereas cells lacking Slx1 are not (5, 31). Furthermore, phosphorylation of Esc4/Rtt107 is defective in cells lacking Slx4 but not in cells lacking Slx1 (36). Moreover, Slx4 is required for recovery from methyl methanesulfonate (MMS)-induced replisome stalling but Slx1 is not (36). Therefore, at least a subset of the cellular roles of Slx1 and Slx4 appears to be distinct.
Slx4 has been also shown to interact physically with proteins other than Slx1. A genome-wide two-hybrid screen identified the Rad1 endonuclease as an Slx4 interactor (22). Rad1 catalyzes DNA incision on the 5' side of UV-induced lesions and cleaves nonhomologous tails generated by DNA end resection during the SSA mode of DNA repair, responsible for repair of double-strand breaks between repeated sequences (1, 11). However, it is not clear if the endogenous cellular form of Slx4 interacts with Rad1 or if this impacts on the function of either protein. Several groups reported that Slx4 interacts with Esc4/Rtt107 (6, 36, 51). Esc4 was originally identified in a screen for genes that regulate retrotransposition in yeast (40) and in global genome screens for genes required for resistance to MMS (5, 17). Esc4 was subsequently shown to be required for completion of chromosome replication after replisome stalling (36, 37), but it is not yet known how it fulfills this task. Cells lacking Esc4 are hypersensitive to a wide range of agents that cause replisome stalling: camptothecin (CPT; causes S-phase-specific DSBs and replisome collapse) and hydroxyurea (HU; slows replication down by depleting deoxynucleoside triphosphates), as well as MMS, whereas cells lacking Slx4 are not hypersensitive to CPT or HU, suggesting that Esc4 has cellular roles not shared by Slx4. However, esc4 and slx4 cells show a comparable level of hypersensitivity to MMS and are epistatic in this regard, suggesting that they share a similar role in promoting resistance to MMS.
Slx4 becomes phosphorylated in response to a wide range of different types of DNA damage, at all cell cycle stages, and this requires both the Mec1 and Tel1 protein kinases (12). These kinases, the yeast orthologues of ATR and ATM in higher eukaryotes, respectively, belong to the PIKK family of kinases, which also includes DNA-dependent protein kinase (DNA-PK) (45). Mec1 and Tel1 phosphorylate target proteins, including the Chk1 and Rad53 kinases, on Ser/Thr-Gln (S/T-Q) motifs and are critically important regulators of several aspects of the cellular response to DNA damage and stalled replisomes (45). The Slx4 residues phosphorylated by Mec1 and Tel1 and the functional significance of Slx4 phosphorylation are not yet known. Here we report that Slx4 has at least three independent cellular functions that appear to require different Slx4-interacting proteins, and we show that one of these functions is regulated by Slx4 phosphorylation.
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MATERIALS AND METHODS |
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-factor (5 µg/ml). When budded cells accounted for less than 5% of the population, cells were released from arrest by filtration and extensive washing and incubated in YPD for 10 min before addition of MMS (0.05%). After 45 min in MMS, cells were filtered, washed extensively with YPD containing 2.5% (wt/vol) sodium thiosulfate, and incubated in YPD at 30°C. At the times indicated, 1 x 108 cells were removed and fixed in 70% ethanol at 4°C overnight before preparation of chromosomes, exactly as described in the CHEF DRII instruction manual (Bio-Rad). Pulsed-field gel electrophoresis (PFGE) was carried out using a Bio-Rad CHEF DRII apparatus at 14°C in a 1% agarose gel (pulsed-field certified; Bio-Rad) in 0.5x Tris-borate-EDTA for 24 h at 6 V/cm using a 120o included angle with a 6.8- to 158-s switch-time ramp. Gels were stained with 10 µg/ml ethidium bromide for 30 min and washed for 2 min in water before DNA was visualized. Measurement of spontaneous mutation frequency. The frequency of forward mutations at the CAN1 gene locus was determined by the frequency of appearance of canavanine-resistant colonies that grew on selective minimal medium plates lacking Arg but containing canavanine (60 µg/ml) (for example, see reference 42a). Cultures were grown to stationary phase for 24 h in minimal medium lacking Arg. The OD600 of cultures was measured, and from the same culture, in parallel, approximately 2 x 107 cells were plated onto canavanine plates and 2 x 102 cells were plated onto YPD plates. The colonies were counted after incubation at 30°C for 3 days. To calculate spontaneous mutation frequencies, the number of canavanine-resistant colonies per ml of culture was divided by the number of CFU (on YPD) per ml of culture. Mutation frequencies represented the average from three independent triplicate experiments, and the relative frequency was calculated from the increase or decrease in mutation frequency in comparison to the wild-type strain.
DNA combing.
Wild-type and slx4 cells containing the human nucleotide transporter hENT1 on a centromeric plasmid (pRS415) and seven copies of the herpes simplex thymidine kinase gene were synchronized in late G1 for 2.5 h with 2 µg/ml -factor (GenePep). Cells were released in S phase with 50 mg/ml Pronase (Calbiochem) in the presence of 30 µg/ml bromodeoxyuridine (BrdU) and 0.05% MMS. After 60 min, MMS was quenched with sodium thiosulfate and cells were resuspended in fresh medium containing 30 µg/ml BrdU. Genomic DNA was extracted in LMP agarose plugs (800 ng/plug) and was stained with YOYO-1 (Molecular Probes). DNA was resuspended in 50 mM morpholineethanesulfonic acid, pH 5.7, to a final concentration of 150 ng/ml. DNA fibers were stretched on silanized coverslips as described elsewhere (29) and were denatured for 25 min in 1 N NaOH. BrdU was detected with a rat monoclonal antibody (clone BU1/75; AbCys) and a secondary antibody coupled to Alexa 488 (Molecular Probes). DNA molecules were counterstained with an antiguanosine antibody (Argene) and an anti-mouse IgG coupled to Alexa 546 (Molecular Probes). Images were recorded with a Leica DM6000B microscope coupled to a CoolSNAP HQ charge-coupled device camera (Roper Scientific) and were processed as described previously (33). MetaMorph v6.2 (Universal Imaging Corp.) was used to measure BrdU signals and DNA fibers, and statistical analysis was performed with Prism 4 (GraphPad Software, Inc.).
Antibody production. All peptides were synthesized by Graham Bloomberg, University of Bristol. Antibodies against phosphorylation sites in Slx4 were raised at the Scottish Antibody Production Unit (Lanarkshire, Scotland) by immunizing sheep with the following peptides coupled to keyhole limpet hemocyanin: AQKSPMpTQETTKN (phospho-Thr-72), LDNQESpSQQRLWT (phospho-Ser-289), and VNFLSLpSQVMDDK (phospho-Ser-329), where pS and pT are phospho-Ser and phospho-Thr, respectively. Antibodies were purified by affinity chromatography on CH-Sepharose to which the phosphopeptide immunogen had been covalently coupled. Phospho-specific antibodies were used at a final concentration of 4 µg/ml in the presence of 50 µg/ml non-phospho peptide in a Western blot analysis.
Miscellaneous methods. Western blotting of extracts prepared by the trichloroacetic acid lysis method (38), preparation of native cell extracts and Myc-Slx4 immunoprecipitation, measurement of SSA induced by a run of trinucleotide repeats placed between direct repeats (13), measurement of HO-induced SSA (16) and fluorescence-activated cell sorter (FACS) analysis (12) were all carried out as described previously.
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RESULTS |
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cells and in sgs1 cells (46), but not in cells lacking Slx1 (Fig. 1A).
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-factor arrest into S phase in the presence of MMS and then washed free of MMS. FACS analysis (Fig. 1B) revealed that, like wild-type cells, slx4 cells had an apparently 2C DNA content by 4 h postrecovery, indicating that the majority of the chromosomes in these cells had been replicated. More than 90% of slx4 cells were viable at this time point (data not shown). DNA replication during recovery from MMS-induced replisome stalling in slx4 cells was investigated further by DNA combing (28, 29). Cells were released from G1 into MMS-containing medium in the presence of BrdU. After 45 min, MMS was washed out and cells were allowed to recover for 90 min or 130 min in fresh medium in the continued presence of BrdU. After combing, chromosome fibers were stained with anti-DNA antibodies (red) or anti-BrdU antibodies (green) (Fig. 1C). This analysis revealed striking defects associated with loss of Slx4. BrdU tracks were almost 50% shorter in slx4 cells than in wild-type cells 90 and 130 min after recovery (Fig. 1C and D), suggesting that DNA replication during recovery from MMS is slower in slx4 cells. Even so, at 130 min after release of cells from MMS, more than 90% of the stalled replisomes had resumed DNA replication in the absence of Slx4 (Fig. 1C and E). However, unreplicated gaps were observed in around 26% of the fibers in slx4 cells, compared with around 8% in wild-type cells (Fig. 1E). These data are reminiscent of the defects seen in cells lacking the cullin Rtt101, which is also required for recovery from DNA alkylation damage (45). In rtt101 cells, unreplicated gaps were detected in 22% of chromosome fibers, compared with only 2% in wild-type cells, and CldU tracks were 50% shorter as observed in slx4 cells (45). We also noticed that DNA fibers isolated from slx4 cells were significantly shorter than those isolated from wild-type cells (Fig. 1E), presumably because they are more fragile and break during the combing process due to the persistence of unreplicated chromosomal gaps and stalled replisomes. Taken together, these data indicate that although cells lacking Slx4 restart DNA synthesis when replisomes stall, DNA replication is slower than normal. Also, at the latest time point examined, chromosomes from slx4 cells are left with unreplicated gaps. This would account for the inability of chromosomes to enter pulsed-field gels (Fig. 1A). SLX4 interacts with genes involved in error-free DNA damage bypass genes. One possible explanation for the recovery defect described above could be that Slx4 promotes repair of DNA alkylation damage. Epistasis analysis with mutants defective in base excision repair (BER), the principal pathway for repair of DNA alkylation damage, was carried out. Cells lacking both SLX4 and methyladenine DNA glycosylase (MAG1), a BER factor that cleaves methylated bases to leave an abasic site in DNA, are more sensitive to MMS than either of the respective single mutants (Fig. 2A). Similar results were obtained with APN1 (Fig. 2A), which is also required for BER. We conclude that BER or nucleotide excision repair (NER) (data not shown) is unlikely to be the major function of SLX4.
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or rad6 cells to MMS (Fig. 2B, lower panel). Therefore SLX4 has an epistatic relationship with genes that regulate DNA damage bypass.
There are two major pathways in cells for bypassing DNA lesions: TLS and error-free bypass (47). TLS occurs by transient recruitment to stalled replisomes of specialized TLS DNA polymerases that, unlike the replicative polymerases 
and 
, can replicate across DNA lesions (47). Rad6/Rad18-dependent mono-ubiquitination of PCNA at Lys164 is thought to recruit TLS polymerases to stalled replisomes. Error-free bypass requires the addition of further ubiquitin moieties to mono-Ub PCNA, and this is catalyzed by the Rad5/Mms2/Ubc13 complex (47). We sought to determine which of these bypass pathways is regulated by SLX4. The major error-prone translesion polymerase in budding yeast is polymerase 
(Pol ), comprising Rev3, the catalytic subunit, and Rev7 (35, 47). The MMS sensitivity of slx4 rev3 cells was greater than that of the single mutants (Fig. 2C), suggesting that SLX4 is not involved in error-prone translesion synthesis and pointing instead to error-free bypass. Consistent with this, slx4 mms2 double mutants were not more sensitive to MMS (Fig. 2D) than the most sensitive single mutants. These data suggest that the inability of slx4 cells to complete DNA synthesis after replisome stalling may be due to an inability to carry out error-free bypass.
Translesion synthesis by Pol is responsible for 50 to 75% of spontaneous cell mutations, and so deletion of Rev3 decreases cell mutation frequencies (26). In contrast, mutations in error-free bypass factors increase spontaneous mutation frequency because of compensatory increases in TLS (4, 41). We reasoned that if Slx4 regulates error-free bypass, then Slx4 deficiency should increase the frequency of spontaneous mutation. Therefore, the frequency of forward mutation in the CAN1 gene, which gives rise to canavanine resistance, was measured. In the genetic background used in this study, deletion of REV3 decreased spontaneous mutation frequency, whereas disruption of UBC13 caused an approximately 8.5-fold increase in mutation frequency (Table 2), consistent with previous reports (4). Cells lacking Slx4 showed an approximately fivefold increase in the frequency of mutation, and this was abrogated by deletion of Rev3 (Table 2). Therefore, cells lacking Slx4 have a "mutator" phenotype that is caused by increased translesion synthesis, like cells lacking error-free bypass factors. This is consistent with, but does not prove, a role for Slx4 in error-free DNA damage avoidance.
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rad1 cells were not more hypersensitive to MMS than wild-type cells, and slx1 rad1 slx4 cells were not more hypersensitive to MMS than slx4 cells (Fig. 3B). Therefore, the role of Slx4 in promoting cellular resistance to MMS, which may involve regulating error-free bypass, appears to be independent of Slx1 and Rad1.
Slx4 regulates single-strand annealing.
The observation that Slx4 interacts physically with Rad1-Rad10 in cells prompted us to test the role of Slx4 in Rad1-Rad10-dependent processes. Rad1-Rad10 is involved in NER, but as slx4 cells are not hypersensitive to UV (data not shown), Slx4 does not appear to be involved in NER. Rad1-Rad10 also plays an important role during the SSA mode of DSB repair that can repair a DSB between repeated sequences (Fig. 3D). After resection of the ends, complementary strands of the homologous regions flanking the DSB can anneal, producing an intermediate that has two nonhomologous 3'-ended tails. Rad1 cleaves the 3' single-strand tails, and the resulting nicks are sealed by ligation (1, 11). Thus, SSA results in deletion of one copy of the repeat plus the sequence located between the repeats.
Zakian and colleagues reported a system for measuring SSA repair of DSBs induced by a hairpin placed between two direct repeats (Fig. 3C, top) (13). When a run of 250 tandem CTG trinucleotide repeats (TNRs) and the URA3 gene are placed between two direct repeats (Fig. 3C, top), the TNRs form a hairpin that causes replisome stalling and DSB formation. These breaks are repaired by SSA in a manner dependent on RAD1and RAD52, resulting in loss of the URA3 marker from between the direct repeats and, consequently, cells become resistant to 5-fluoroorotic acid (5-FOA); no marker loss was observed in the absence of CTG repeats (13). As shown in Fig. 3C (bottom), a high frequency of URA3 loss is observed in when TNRs (CTG-250) are located between the direct repeats, but not in the absence of TNRs (CTG-0). Disruption of Slx4, however, like disruption of Rad1 (13), causes a severe reduction in SSA in this assay (Fig. 3C). However, disruption of MMS2 that regulates error-free bypass did not have a statistically significant effect on marker loss (Fig. 3C, bottom), and this demonstrates that marker loss in this assay is not due to sister strand slippage caused by template switching at the TNRs. Furthermore, disruption of Slx1 had no effect on SSA (Fig. 3C, bottom). This argues that Slx4 regulates Rad1-dependent SSA independently of Slx1 and independently of its role in error-free bypass.
We tested the effect of disruption of Slx4 in a different assay for SSA that did not rely on TNRs for formation of DSBs. Instead, the HO endonuclease was induced to create a single DSB between 205-bp repeated segments of the URA3 gene (Fig. 3D) (43). Induction of a GAL::HO gene efficiently induced SSA in wild-type cells and created deletion products that could be monitored by Southern hybridization (Fig. 3E), and it is well established that this requires Rad1-Rad10 (11). Strikingly, disruption of Slx4 in this background also abolishes SSA. Taken together, the data in this section indicate that Slx4 interacts with Rad1-Rad10 and promotes repair of DSBs by SSA.
Phosphorylation of Slx4 by Mec1 and Tel1 regulates SSA. DNA damage induces Mec1/Tel1-dependent phosphorylation of Slx4 that is independent of Rad53 and Chk1 (12). Mec1/Tel1, ATR/ATM, and DNA-PK all have identical specificity in vitro, and even though DNA-PK is not found in yeasts, it phosphorylates the same motifs in vitro in Slx4 as Mec1 and Tel1 (45). Since DNA-PK is readily available, we determined the residues in Slx4 phosphorylated by DNA-PK in vitro as a starting point to ultimately find the residues phosphorylated in vivo by Mec1/Tel1. Incubation of recombinant His6-Slx4 with DNA-PK resulted in phosphorylation of His6-Slx4 with a stoichiometry of approximately 6 moles of phosphate per mole of Slx4 (data not shown). The sites phosphorylated in Slx4 were identified by a combination of mass spectrometry and Edman degradation (data not shown). These analyses revealed that Slx4 is phosphorylated in vitro by DNA-PK on the following six residues: Thr72, Thr113, Ser289, Thr319, Ser329, and Ser355, and all of these conformed to the S/T-Q consensus sequence (Table 3).
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tel1 cells. Phosphorylation of these residues occurred at wild-type levels in cells lacking either Mec1 or Tel1 and in cells lacking both Rad53 and Chk1 (data not shown). Thus, both Mec1 and Tel1 phosphorylate Slx4 on several S/T-Q motifs after DNA damage in vivo.
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cells, the Slx4 mutants were expressed at similar levels to wild-type Slx4 (data not shown).
We tested if the Slx4 phosphorylation site mutants could rescue the MMS hypersensitivity of slx4 cells. Cells expressing Slx4-Mut3 (data not shown) or Slx4-Mut6 (Fig. 5A) were no more sensitive to MMS than cells expressing wild-type Slx4. However, it was possible that phosphorylation of Slx4 by Mec1/Tel1 at S/T-Q sites other than those mutated in pSLX4-MUT6 may have provided resistance to MMS. This is unlikely, since mutation of 16 of the total 18 S/T-Q motifs in Slx4 had no effect on the ability of Slx4 to rescue the MMS hypersensitivity of slx4 cells (data not shown). Thus, it is highly unlikely that Mec1/Tel1-dependent phosphorylation of Slx4 is required for its ability to promote resistance to MMS or, therefore, error-free bypass. However, we reasoned that phosphorylation of Slx4 may affect an aspect of its function other than cellular resistance to MMS, such as SSA or the ability to maintain cell viability in the absence of Sgs1. Therefore, we investigated the role of phosphorylation in regulating these aspects of Slx4 function.
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and slx4 are inviable but can be kept alive by a low-copy-number [pSGS1-URA3-ADE] plasmid expressing SGS1. These cells are sensitive to killing by FOA, which is converted to toxic intermediates by Ura3, since cells cannot lose the SGS1 plasmid (30, 31). sgs1 slx4 ade2 ade3 [pSGS1-URA3-ADE] cells (strain NJY561) (5) were transformed with the following HIS3 plasmids: empty plasmid (pRS413), pSLX4, or pSLX4-MUT6. Cells were then restreaked to YPD plates with or without 5-FOA. Cells transformed with empty plasmid (pRS413) are FOA sensitive and red (since they cannot lose the ADE3 gene). Introduction of SLX4 on a low-copy-number plasmid allowed sgs1 slx4 [pSGS1-URA3-ADE] cells to grow on FOA (Fig. 5B) and caused red-white sectoring on low-adenine plates (data not shown), since cells could now lose the SGS1-URA3 plasmid. Introduction of pSLX4-MUT6, like wild-type SLX4, resulted in FOA resistance (Fig. 5B). This indicates Slx4 phosphorylation is not essential for viability in sgs1 cells. We next tested the impact of Slx4 phosphorylation on SSA. Whereas wild-type Slx4 could rescue the decrease in SSA observed in slx4 cells, the Slx4-Mut3 or Slx4-Mut6 phospho site mutants could not (Fig. 5C). This is consistent with previous observations that cells lacking Mec1 show reduced SSA (24). Taken together, the data in this section indicate that Mec1/Tel1-mediated phosphorylation of Slx4 regulates SSA but not cell viability in the absence of Sgs1 or cellular resistance to MMS. |
DISCUSSION |
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cells and is not required for cellular resistance to MMS.
The inability of slx4 cells to recover from MMS-induced replisome stalling (Fig. 1A) (36) prompted us to investigate if DNA replication could resume in these cells during recovery or whether problems arose instead in completing S phase. We found that although DNA replication can resume after replisome stalling in cells lacking Slx4 (Fig. 1B), DNA synthesis is slow and there is a major defect in the completion of replication, resulting in unreplicated gaps in the genome (Fig. 1C to E). Precisely why slx4 cells have problems in completing DNA replication is not yet clear. However, in this study we found that SLX4 interacts with genes involved in error-free DNA damage bypass, and several lines of evidence suggest that Slx4 might regulate this process. Firstly, Slx4 is epistatic with error-free bypass genes in terms of MMS hypersensitivity (Fig. 2). Secondly, cells lacking a known error-free DNA damage bypass factor, Mms2, have a similar recovery defect to that seen in slx4 cells (data not shown). Thirdly, the spontaneous mutation frequency is elevated in slx4 cells, caused by a compensatory increase in TLS. This also occurs in known error-free bypass mutants (Table 2) and probably masks the real severity of the consequences of disrupting Slx4, especially given the additive effect of disrupting REV3 in slx4 cells (Fig. 2C).
Error-free bypass is thought to involve template switching initiated by unpairing and annealing of the blocked nascent strand with the intact nascent sister strand, but the mechanisms and gene products involved are not clear. After replication past the blockage, the nascent strands would unpair again and reanneal with the parental strands (20, 39). There is experimental evidence to support template switching at blocked replisomes in yeast (53), but since the mechanisms involved are unclear, as are many of the relevant genes, it is difficult to speculate about the potential role of Slx4. Template switching should involve the action of helicases and nucleases, and it is possible that Slx4 recruits one or more catalytic activities to replisomes blocked by DNA lesions. In this light, it is interesting that Slx4 interacts with Slx1 that in vitro cleaves structures that resemble those that may arise during error-free bypass. However, Slx1 is not required for cellular resistance to MMS. Genome-wide analysis revealed the Rad1 subunit of the Rad1-Rad10 nuclease as a potential Slx4 interactor, and in this study we demonstrated that cellular Slx4 interacts with Rad1-Rad10 (Fig. 3A). However, rad1 cells recover normally from MMS (data not shown) and are not MMS sensitive (Fig. 3B). It is unlikely, therefore, that Rad1 and Slx1 function redundantly in error-free bypass since slx1 rad1 cells are not hypersensitive to MMS (Fig. 3B).
Slx4 interacts with Esc4, which is also required for recovery from replisome stalling (36, 37), but unlike Slx4, disruption of Esc4 causes sickness but not lethality in sgs1 cells (51). Cells lacking Esc4 are hypersensitive to CPT and HU (44), whereas cells lacking Slx4 are not (data not shown), suggesting that Slx4 and Esc4 have distinct functions. However, esc4 and slx4 cells show a comparable level of hypersensitivity to MMS and are epistatic in this regard (6, 36). In addition, Slx4 is required for phosphorylation of Esc4 by Mec1 (36), indicating that the Slx4-Esc4 interaction is functionally important. It remains to be determined how the association with Esc4 impacts on Slx4 function in promoting resistance to MMS. It will be of particular importance to examine the ability of these proteins to associate with stalled replisomes and how this is regulated.
Rad1-Rad10 is essential for SSA (1, 11, 13), and because we found that Slx4 interacts with this complex, we tested if Slx4 regulates SSA. Indeed, slx4 cells showed a severe reduction in Rad1-Rad10-dependent SSA in two different assays (Fig. 3). It is not yet clear why deletion of Slx4 has such a profound effect on SSA but could reflect a role in Rad1-mediated removal of nonhomologous tails, in annealing of the repeats, or in directing new DNA synthesis prior to ligation of the ends. Since the Slx4-Slx1 complex also cleaves flaps and branched structures, it is tempting to speculate that Slx4 recognizes and binds to these structures in a manner that somehow facilitates cleavage by associated enzymes. However, Slx1 does not appear to be involved in SSA, and the Slx4-Slx1 and Slx4-Rad1-Rad10 complexes appear to be distinct. It is not, therefore, clear how the different complexes recognize the appropriate lesions. Biochemical analysis of these complexes and the identification of separation-of-function mutants may help to elucidate this problem.
In this study we identified six residues in Slx4 phosphorylated by DNA-PK in vitro. Phospho-specific antibodies demonstrated clearly that at least three of these sites—Thr72, Ser289, and Ser329—become phosphorylated in vivo in response to DNA damage but not in cells lacking both Mec1 and Tel1 kinases. Mutation of these three sites in Slx4 phosphorylated in vivo after DNA damage does not affect cell viability in the presence of MMS or viability in cells lacking Sgs1 but does inhibit Rad1-dependent SSA. This suggests that the role of Slx4 in SSA is distinct from its role in promoting cell viability in the absence of Sgs1 and in promoting resistance to MMS (Fig. 6).
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Based on the data in this study, we postulate the existence of at least two mutually exclusive, functionally distinct Slx4-containing complexes (Fig. 6A). Esc4-Slx4-Slx1 protects cell viability in the absence of Sgs1 but is not required for SSA. The Slx4-Rad1-Rad10 complex, on the other hand, promotes SSA. The two Slx4-containing complexes appear to be distinct, in that deletion of RAD1 is not synthetically lethal with deletion of SGS1 (48) but deletion of SLX1 is (31). In addition, Rad1-Rad10 but not Slx1 is required for SSA (Fig. 3C and E). The ability of Slx4 to promote cellular resistance to MMS probably involves the Esc4-Slx4-Slx1 complex: even though Slx1 is not required for recovery from MMS (Fig. 1A), Esc4 and Slx4 are epistatic with regard to MMS hypersensitivity (36). Alternatively, one or more as-yet-unidentified Slx4-interacting proteins may fulfill this task. It will be important to identify such proteins, to examine the dynamics of Slx4-containing complexes and how Slx4 is distributed among them, and to study how Slx4 phosphorylation influences Slx4 function at the molecular level.
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ACKNOWLEDGMENTS |
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S.F. was funded by a predoctoral fellowship from the Medical Research Council (MRC) UK, and work in the Rouse lab is funded by the MRC, the Association for International Cancer Research, and an EMBO Young Investigator Award.
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FOOTNOTES |
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Published ahead of print on 16 July 2007.
Present address: Gurdon/CRUK Institute, University of Cambridge, Tennis Court Rd., Cambridge, United Kingdom.
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