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Previous Article | Next Article 
Molecular and Cellular Biology, March 2006, p. 1617-1630, Vol. 26, No. 5
0270-7306/06/$08.00+0 doi:10.1128/MCB.26.5.1617-1630.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Molecular Biology,1 Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 920372
Received 26 September 2005/ Returned for modification 19 October 2005/ Accepted 6 December 2005
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ABSTRACT |
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INTRODUCTION |
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In addition to their essential functions, the SMC complexes all appear to facilitate DNA repair. De novo cohesin deposition in the vicinity of double-strand breaks (DSBs), which enhances their repair, has recently been demonstrated (56, 60). Interestingly, local removal of cohesin from damage sites has also been suggested to be important for normal repair (39). Condensin has been implicated in interphase DNA damage responses, where it is required for repair of UV-induced DNA damage and also for the activation of the replication checkpoint kinase Cds1 (4). Further, a subunit of condensin has been found to associate with DNA ligase IV, supporting a direct role in DNA repair (49). Finally, Rad18 (Smc6) was identified in screens for radiation-sensitive mutants of fission yeast and, subsequently, mutations in a number of the subunits of the SMC5/6 complex have been found to confer DNA repair defects (17, 19, 30, 34, 44). Several studies have implicated the Smc5/6 complex in the process of DSB repair via homologous recombination in vegetative cells (19, 30, 34, 36, 44). The Smc5/6 complex has also been shown to play an important role in meiotic recombination and the subsequent segregation of chromosomes at meiosis I (44). A recent study in budding yeast suggested that unresolved recombination structures between the repetitive sequences on chromosomes, i.e., telomeres and rRNA genes, accounts for some of the DNA segregation defects and cell death seen in SMC5/6 mutants (59).
The chromatin-associated activities of the SMC heterodimers described above are dependent on several non-SMC subunits (31, 40). These subunits have been well described and characterized for the cohesin and condensin complexes (31, 40). However, the subunit composition and architecture of the SMC5/6 complex has only recently begun to be addressed. Four non-SMC elements, Nse1 to -4, have been identified that are conserved across species at the primary sequence level (31, 40). Nse1 and Nse2 are intriguing, since they contain zinc fingers of the RING and PIAS family, respectively (34). These domains suggest that Nse1 and Nse2 act as E3 ligases for ubiquitylation and sumoylation of various protein targets, respectively (16, 18, 23, 34, 58, 64). Indeed, Nse2 of fission yeast and its homologues in budding yeast and humans (MMS21/NSE2) have recently been shown to stimulate the sumoylation of certain DNA repair factors (3, 47, 66). The physiological role of these modifications remains to be determined. Nse3 contains a MAGE homology domain, which is found in a large family of mammalian proteins that are highly expressed in tumors (6, 44, 54). The function of the MAGE domain is currently unknown, but members of the family play roles in cell growth and transcriptional regulation (6). Nse4 does not contain any domains predictive of a particular function (36, 44, 54).
In addition to the four well-conserved non-SMC subunits (Nse1 to -4), the budding yeast SMC5/6 complex contains two other essential subunits, YML023c and KRE29, which are not conserved at the primary sequence level in other species (20, 66). To test for the presence of functional counterparts of YML023c and KRE29, we have undertaken a comprehensive mass spectrometric analysis of proteins copurifying with fission yeast Smc5 and Nse4. This approach has led to the identification of two intriguing factors we call Nse5 and Nse6. Surprisingly, unlike YML023c and KRE29, Nse5/6 are nonessential. Nse5/6 form an obligate heterodimer, which appears to specifically facilitate the DNA repair roles of the Smc5/6 complex. KRE29 and Nse6 are both 
50-kDa proteins of the ARM/HEAT repeat family, which includes subunits/regulators of the cohesin and condensin complexes (31). That both fission and budding yeast Smc5/6 complexes are octameric suggests that factors equivalent to Nse5/6 and YML023c/KRE29 await identification in other species. Biochemical studies of the Nse5/6 heterodimer show that it binds directly to Smc5/6, independently of Nse1 to -4. This is the first reported reconstitution of the octameric Smc5/6 complex and includes revisions to the current architectural model of the complex (54).
Via genetic arguments, others and we recently proposed a role for Smc5/6 in the mitigation of replication fork-associated damage (36, 44). In the present study, we find that Nse5/6 mutants display a high rate of spontaneous DNA damage and are hypersensitive to replication fork blockage induced by DNA alkylation or UV lesions. This hypersensitivity is dependent on intact homologous recombination repair machinery (HRR) and is partially rescued by overexpression of the bacterial Holliday junction resolvase RusA. Consistently, the viability of Nse6 mutants depends on the activity of Mus81 and Rqh1 (hBLM), factors that resolve or prevent the formation of Holliday junctions (7, 12, 13).
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MATERIALS AND METHODS |
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Immunoblotting. Proteins denatured in SDS sample buffer (1% SDS, 5% glycerol, 0.1% bromophenol blue, 40 mM Tris-HCl [pH 6.8], 1/10 [vol/vol] ß-mercaptoethanol) were resolved on 8 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred on Immobilon-P membrane (Millipore, Billerica, MA), blocked in 5% milk in Tris-saline buffer with 0.3% Tween 20, and probed with mouse monoclonal antibodies raised against the myc (9E10; BABCO, Richmond, CA), HA (12CA5; BABCO), FLAG (anti-FLAG M2; Sigma-Aldrich, Saint Louis, MI), or His6 (6xHis antibody; BD Biosciences, Palo Alto, CA) epitopes. The peroxidase antiperoxidase reagent (PAP; Sigma-Aldrich) was used for detection of TAP-tagged proteins. After amplification by probing with a secondary horseradish peroxidase-conjugated rabbit anti-mouse antibody, the protein signal was revealed by using ECL reagent (Pierce, Rockford, IL) on autoradiography films (Genesee, San Diego, CA).
Immunoprecipitation of endogenous Schizosaccharomyces pombe proteins. Endogenously TAP-tagged S. pombe cells were lysed in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.2% Nonidet P-40, 1x Complete protease inhibitor mix EDTA-free [Roche, Indianapolis, IN], and 1 mM phenylmethylsulfonyl fluoride [PMSF]). Soluble lysates cleared by centrifugation were mixed with immunoglobulin G (IgG)-Sepharose (Pfizer, New York, NY), followed by incubation for 2 h at 4°C with rotation. Beads were then washed three times in lysis buffer, resuspended, and boiled in SDS sample buffer.
Expression of recombinant S. pombe proteins and interaction assays in insect cells. DNA sequences encoding the open reading frames of Nse1 to -6 and Smc5/6 were amplified by PCR from a S. pombe cDNA library using Pfu Turbo (Stratagene, La Jolla, CA) and cloned in frame into the His6 tag-containing Bac-to-Bac baculovirus expression vector pFastBac HT (Invitrogen, Carlsbad, CA). The sequence encoding the His6 tag in the N terminus was then replaced in frame by a DNA fragment containing either a hemagglutinin (HA), a FLAG tag, or a cassette containing the glutathione S-transferase (GST) tag and a cleavage site for the PreScission protease (protein sequence LEVLFQGP) between GST and the open reading frame. These vectors were then used to transform DH10Bac Escherichia coli cells according to the manufacturer's instructions (Invitrogen) to allow their transposition into a shuttle vector and generate recombinant bacmids.
For recombinant protein expression, Sf9 insect cells were grown in Ex-Cell 401 w/L-glutamine insect cell medium (JRH Biosciences, Lenexa, KS) supplemented with 10% (vol/vol) penicillin-streptomycin-glutamine mix (Invitrogen). Sf9 cultures were supplemented with 2% super calf serum (Gemini, Woodland, CA) during infections only. To obtain recombinant baculoviruses, each bacmid was transfected into Sf9 insect cells by using Effectene transfection reagent (QIAGEN, Valencia, CA) according to the manufacturer's instructions, and baculoviral supernatants were collected after incubation of transfected Sf9 cells for 5 days at 28°C. These primary baculoviral stocks were amplified by two rounds of Sf9 infections to produce high-titer virus stocks. Protein expression was checked by immunoblotting infected Sf9 whole-cell lysates.
To test for direct interaction between recombinant proteins by FLAG purification, 108 Sf9 cells were coinfected in suspension with a multiplicity of infection of 1 to 10 of each of the indicated baculoviral stocks in 2% super calf serum, followed by incubation for 45 to 48 h in a 28°C shaker. Cells were then collected by centrifugation and lysed in 10 ml of HEMG-500 buffer (25 mM HEPES [pH 7.4], 500 mM NaCl, 0.5 mM EDTA, 1.5 mM MgCl2, 10% glycerol, and 0.05% Nonidet P-40, supplemented with 1x complete EDTA-free protease inhibitor mix [Roche] and 1 mM PMSF) for 10 min on ice. After one freezing in liquid nitrogen, cells were quickly thawed and sonicated with a Braun-Sonic 2000 sonicator (B. Braun Instruments) for three pulses of 15 s at an output of 70. Lysates were cleared by high-speed centrifugation at 14,000 rpm for 20 min. Soluble fractions were incubated with 100 ml of anti-FLAG M2 affinity gel (Sigma-Aldrich) prewashed in HEMG-500 buffer. Protein complexes were allowed to bind the resin for 2 h at 4°C with rotation before being washed three times with 1 ml of HEMG-500 buffer and resuspended in ß-mercaptoethanol-free SDS sample buffer (125 mM Tris-HCl [pH 6.8], 4% SDS, 20% [vol/vol] glycerol, 0.004% bromophenol blue). Proteins were denaturated by a 3-min boil, the affinity gel was pelleted by centrifugation, and the supernatant was transferred to a new tube, supplemented with 1/10 (vol/vol) ß-mercaptoethanol, and reboiled for 3 min before being subjected to immunoblotting. This procedure avoids elution of the IgG heavy chain, which masks proteins with a similar molecular weight during immunoblotting. For GST-tagged protein purification, the same method was applied by using GST lysis buffer instead of HEMG-500 (1x phosphate-buffered saline complemented to 500 mM NaCl, 1 mM dithiothreitol, 1% Triton X-100, and 10% glycerol and supplemented with 1x complete EDTA-free protease inhibitor mix [Roche] and 1 mM PMSF). Glutathione-Sepharose 4B beads (GE Healthcare) were used to immunoprecipitate the GST-tagged bait protein. After incubation and washes as described above, immunoprecipitated proteins were either resolved on SDS-PAGE and subjected to Western blotting ("pull-down" samples) or else cleaved off the GST tag by using a GST-PreScission protease fusion (GE Healthcare). To this end, 20 mg of PreScission protease was added to the beads in GST lysis buffer, and protease-mediated cleavage was allowed to proceed for 4 h at 4°C with rotation. After incubation, the eluate-containing supernatant ("eluate" sample) was separated from the pellet containing the beads, the GST peptide, and the GST-PreScission fusion protein by centrifugation, and specifically eluted proteins were analyzed by Western blotting as described above.
TAP purification and mass spectrometry.
Proteins associating with Smc5, Nse4, and Nse6-TAP were identified by MudPIT using established methods (34, 35, 44, 63). Briefly, cells (50 g [wet weight]) expressing TAP-tagged proteins from their genomic loci were frozen in liquid nitrogen and lysed by using a motorized mortar and pestle (Retsch) in buffer A (50 mM Tris [pH 8]; 150 mM NaCl; 2 mM EDTA; 10% glycerol; 0.2% Nonidet P-40; 5 µg each of leupeptin, pepstatin, and aprotinin/ml; 1 mM PMSF). TAP-tagged proteins were purified from clarified lysates as described, except we used only the first step, omitting the secondary purification on calmodulin beads (51). The final eluate was precipitated with trichloroacetic acid (25% [vol/vol]) for 1 h on ice. The precipitate was centrifuged (Eppendorf) at a relative centrifugal force of 16. The pellet was washed twice with acetone (–20°C) and air dried. Trichloroacetic acid precipitates from the TAP purifications were resuspended in digestion buffer (8 M urea, 100 mM Tris-HCl [pH 8.5]) and digested by the sequential addition of Lys-C and trypsin proteases as previously described (33). The digests were then analyzed by multidimensional microscale liquid chromatography followed by mass spectrometry using either a Finnigan LCQ Deca ion trap mass spectrometer or Finnigan LTQ linear ion trap mass spectrometer. Multidimensional chromatography was performed online according to the method of MacCoss et al. (32). The collection of resulting spectra was then searched against a database of S. pombe open reading frames obtained from PombeDB using the SEQUEST algorithm (15). Peptide identifications were organized and filtered by using the DTASelect program (57). Filtering criteria for positive protein identifications were the identification of two unique, half or fully tryptic peptides with Xcorr values of greater than 1.8 for +1 spectra, 2.5 for +2 spectra, and 3.5 for +3 spectra and a 
Cn value greater than 0.08.
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RESULTS |
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Architecture of the octameric Smc5/6 complex. We have shown that Nse5 and Nse6 interact with the Smc5/6 complex in vivo. However, the approaches used thus far do not allow us to determine what direct protein-protein contacts (if any) are made with other elements of the complex. To address this issue, we expressed various combinations of the Smc5/6 complex proteins in an insect cell system. In each experiment, the "bait" protein was tagged with a FLAG epitope and tested for its interaction with all of the other HA- or His6-tagged subunits by immunoprecipitation and immunoblotting. None of the HA- or His6-tagged subunits was pulled-down by the anti-FLAG affinity gel alone (Fig. 2A and data not shown). Using this system, we initially found that Nse1, Nse3, and Nse4 form a stable heterotrimer and that Nse2 interacts strongly with Smc5 (data not shown). These observations correlate well with those reported recently (54).
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Since direct contacts between Smc5 and Nse1, Nse3, or Nse4 have not previously been described, we wanted to further dissect the interactions observed in Fig. 2B. Copurification of epitope-tagged Nse1 to -4 with immunoprecipitated FLAG-Smc5 was analyzed by immunoblotting (Fig. 2D). Nse1 alone does not interact with Smc5, whereas Nse3, Nse4, and Nse2 all show affinity for Smc5 (Fig. 2D, lanes 4, 6, 8, and 14, respectively). When Nse1 and Nse3 are coexpressed, a small amount of Nse1 is found to copurify with FLAG-Smc5 (Fig. 2D, lane 10). When Nse1, Nse3, and Nse4 are expressed together, they show robust copurification with FLAG-Smc5 (Fig. 2D, lane 12). Finally, Nse1 to -4 all copurify with FLAG-Smc5 (Fig. 2D, lane 16). These data show that the Nse1/3/4 heterotrimer and Nse2 bind Smc5 independently of each other. The interaction of Nse1 with Smc5 is likely bridged via Nse3 and Nse4.
Since Nse5/6 did not interact directly with Nse1 to -4, we tested their interaction with Smc5/6. We expressed FLAG-Nse5, HA-Nse6, and GST-Smc6 in insect cells, prepared extracts and precipitated GST-Smc6 on GSH-Sepharose. Nse5, Nse6, and the Nse5/6 dimer showed strong affinity for Smc6 (Fig. 3A, lanes 4, 2, and 6, respectively). We also repeated the experiment in the presence of Smc5, the heterodimeric partner of Smc6. As expected, Smc6 bound strongly to Smc5 (Fig. 3B, lane 1). The Smc5/6 heterodimer interacted with Nse5/6 in the same manner as observed for Smc6 alone (Fig. 3B).
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Nse5 and Nse6 are nuclear proteins required for genome stability. The gene encoding Nse6 was initially identified in a screen in which green fluorescent protein (GFP) was randomly fused to fragments of fission yeast genomic DNA, thus generating a library of GFP fusion proteins that could be tested for discrete subcellular localizations (53). The extreme C terminus of Nse6, fused to GFP, was found to localize to the cell cortex (52). Hence, the authors of that study named the gene cor1, for cortex. They also reported that the gene was not essential for vegetative growth. Based on the observations in the present study, we propose the gene be renamed nse6, since this name refers to the function of the native protein. We epitope tagged Nse5 and Nse6 at their genomic loci with the myc epitope. Indirect immunofluorescence demonstrates that Nse5 and Nse6 are both predominantly nuclear proteins, with no significant changes in their staining patterns throughout the cell cycle (Fig. 4A).
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cells lacking the G2 DNA damage checkpoint kinase Chk1 have fewer elongated cells in the population, with a concomitant increase in cells undergoing mitotic catastrophe (Fig. 4B). Further, the nse6 mutation is very sick when combined with a deletion of the master checkpoint regulator, Rad3 (hATR) (50). The nse6 rad3 double mutants grow very slowly and exhibit high levels of mitotic catastrophe with irregular cell size/shape (Fig. 4B). Together, these observations suggest that Nse5/6 are important to prevent and/or repair spontaneous DNA damage. Nse6 mutants, as observed for hypomorphic mutants of other components of the Smc5/6 complex, display aberrant mitoses upon recovery from UV irradiation (Fig. 4B, right panels; see, for example, reference 19). A common aberration is the "stretching" out of DNA along the axis of the mitotic spindle, indicating a defect in the resolution of chromosomes.
The presence of spontaneous and induced DNA damage can be monitored indirectly in cells that express a fusion protein of the HRR factor Rad22 with YFP (14, 42). Rad22-YFP is recruited to induced DNA DSBs and also to sites of presumptive replication fork collapse (14, 42). In wild-type cells, Rad22-YFP foci are observed in a low 5% of cells (Fig. 4C) (14, 42). Strikingly, Rad22-YFP foci were found in 38% of nse6 cells (Fig. 4C). This suggests that, consistent with the observed DNA damage checkpoint activation, nse6 cells have a greatly increased level of spontaneous DNA damage.
Nse5 and Nse6 function interdependently to facilitate DNA repair. The essential Smc5/6 complex plays undefined roles in chromosome transmission and DNA repair (29, 31). To date, the repair roles of Smc5/6 have been studied by using hypomorphic "separation of function" alleles of components of the Smc5/6 complex (see, for example, references 3, 19, 34, 36, 44, and 54). These studies have shown that the Smc5/6 complex facilitates the repair of various forms of DNA damage resulting from UV irradiation, gamma irradiation, alkylating agents and hydroxyurea. We, therefore, tested the sensitivity of cells lacking Nse5 and Nse6 to these genotoxic agents. Nse5/6 mutants are hypersensitive to UV irradiation and also to the replication blocking agent hydroxyurea. These sensitivities are rescued by reintroducing the nse5/6 genes on a plasmid (Fig. 5A and data not shown). Notably, the Nse5 or Nse6 mutant cells display indistinguishable sensitivities to all DNA damaging agents tested, including UV irradiation (Fig. 5B and data not shown). Furthermore, cells lacking both Nse5 and Nse6 are as sensitive to UV irradiation as either single mutant (Fig. 5B). This observation, together with the ability of Nse5/6 to form a robust dimer in insect cells, strongly suggests that Nse5/6 form a functionally interdependent heterodimer.
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to camptothecin demonstrates the requirement for this pathway to survive camptothecin-induced DNA damage (Fig. 5C). That the Nse6 mutant is not hypersensitive to camptothecin suggests that Nse6 does not play a major role in facilitating Rhp51 in recombination-dependent replication restart. Finally, Nse6 mutants are mildly sensitive to gamma irradiation, which causes DSBs (Fig. 5D). In this situation, Nse6 appears to function in an Rhp51-dependent pathway since nse6 rhp51 double mutants are no more sensitive to gamma irradiation than rhp51 alone (Fig. 5D). The spectrum of DNA damage sensitivities displayed by Nse6 support a major role for Nse5/6 in mitigating damage associated with stalled replication forks, before they collapse to produce one-sided DSBs.
Rhp51-dependent UV-induced toxic structures form in Nse5/6 mutants.
Nse5/6 mutants are most sensitive to agents that can cause replication fork stalling, such as UV, MMS, and hydroxyurea. Unlike the response to DSBs, we observed that Nse6 mutant cells were much more sensitive to UV irradiation than cells with Rhp51 deleted (Fig. 6A). In an otherwise wild-type background, the extreme UV sensitivity of Nse6 mutants cannot only be equated to blocking of replication forks. Nse6 may play a role in the nucleotide excision repair processes that remove UV-induced lesions in G2 phase. However, as we show in the next section, Nse6 certainly plays a major role in UV damage tolerance that is independent of the excision repair pathways. The UV sensitivity of Nse6 mutants is comparable to that of a hypomorphic mutant of Nse2, nse2-1 (Fig. 6A) (34). Due to synthetic lethality, we were unable to perform epistasis analysis between nse6 and nse2-1 or any other hypomorphic component of the Smc5/6 complex (Table 2). Interestingly, deletion of Rhp51 rescues the sensitivity of nse6 and nse2-1 cells to UV at low UV doses (Fig. 6A). This observation suggests that in nse6 and nse2-1 cells UV damage is converted into a toxic structure by the HRR protein Rhp51. A similar epistatic relationship between the rhp51 mutant and smc6-X or rad62-1 (nse4-1) has been reported, but the phenomenon was not explored further (30, 36).
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Nse5/6 facilitate tolerance of DNA damage during replication. Cells undergoing replication can survive UV-induced DNA lesions by removing or bypassing them. In fission yeast, removal of UV-induced DNA lesions is carried out by nucleotide excision repair (NER) and a pathway initiated by the UV dimer endonuclease. These pathways rely on the activity of the Rad13 and Uve1 proteins, respectively (10, 65). Other pathways help cells tolerate damage without removing it, either by replicating through the lesion using translesion polymerases (reviewed in references 28 and 48) or bypassing it via a recombination-dependent mechanism (8, 21, 38).
To clarify the role that Nse6 plays in UV damage tolerance/repair, we constructed double mutants between nse6 and various representatives of the pathways described above. We found that nse6 cells are more sensitive to UV than either rad13 or uve1 single mutants (Fig. 7A). The nse6 rad13 and nse6 uve1 double mutants are more sensitive to UV than the respective single mutants, suggesting that Nse6 functions in both pathways, or in a third pathway that does not involve removal of DNA lesions. Indeed, the nse6 rad13 uve1 triple mutant is profoundly more sensitive to UV than the rad13 uve1 double mutant (Fig. 7B). Nse6, therefore, appears to function in the post-replicative repair (PRR) pathways, which include translesion synthesis polymerases and HRR machinery (reviewed in references 28 and 48). In budding yeast, the Rad18/Rad6 ubiquitin-ligase complex regulates the PRR pathways. However, the nse6 mutant shows additive UV sensitivity when combined with a deletion of Rhp18 (the S. pombe Rad18 homologue [27, 62]) (Fig. 7C). This suggests that Nse6 plays repair roles outside of the PRR pathways.
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mutant are similar to those observed for hypomorphic mutants of the Smc5/6 complex and support a role for Nse5/6 in mitigating replication fork-associated damage as part of the Smc5/6 complex (44). Interestingly, in contrast to hypomorphic mutants of the other essential subunits of the Smc5/6 complex, deletions of Nse5/6 are not synthetic lethal with a deletion of Brc1 (36, 44, 61; data not shown). Therefore, Nse5/6 mutation compromises some, but not all, functions of the Smc5/6 complex, providing a separation of its DNA repair and essential functions.
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Based on the synthetic lethality of mus81 or rqh1 mutants with a deletion of Nse6, we tested the ability of RusA to suppress nse6 mutant phenotypes. We found that expression of catalytically active RusA was able to partially suppress the sensitivity of Nse6 mutant cells to UV irradiation (Fig. 8B). It also slightly improved the growth rate of unirradiated nse6 cells. Catalytically dead RusA, as for the vector control, was unable to rescue the UV sensitivity of the Nse6 mutant (Fig. 8C). Overexpression of Brc1, a protein previously found to partially suppress the damage sensitivities of different Smc5/6 complex mutants, made Nse6 mutants sicker and predictably provided no rescue of their UV sensitivity (Fig. 8C). Together with the genetic interactions, these data suggest that Nse5/6 and possibly the Smc5/6 complex prevent the formation of excessive Holliday junctions or assist in their resolution.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We believe that with the discovery of Nse5/6 the fission yeast Smc5/6 holocomplex has been fully defined. Mass spectrometry and in vivo coimmunoprecipitation data suggest that there is in fact a single complex that contains Smc5/6 and Nse1 to -6. The existence of a single octameric complex is further supported by our studies using an insect cell expression system. These studies have confirmed that stable Nse1/3/4 and Nse2/Smc5 subcomplexes can form independently of each other (54; data not shown). However, a stable interaction between these subcomplexes was not detected in a prior study (54). Using an insect cell system, we have been able to reconstitute the entire octameric Smc5/6 complex in a soluble form. An important finding is that the Nse1/3/4 trimer binds directly to Smc5, independently of Nse2, Nse5/6, and Smc6. Recently, using the yeast two-hybrid assay, a weak interaction detected between Nse2 and Nse3 was suggested to bridge the Nse1/3/4 and Smc5/Nse2 complexes (54). The finding that Nse2 acts as an E3 ligase for the sumoylation of Nse3 could explain the weak interaction detected between these proteins (3). The difference in the architecture of the Smc5/6 holocomplex determined in the present study versus that of a prior study most likely stems from the protein expression systems used. The prior study used a combination of bacterial expression and in vitro transcription or translation (54). In comparison to expression of proteins in insect cells, these systems are more likely to exhibit incorrect protein folding and the absence of certain posttranslational modifications.
Nse5/6 apparently interacts with the Smc5/6 heterodimer through multiple contacts, suggesting an extensive interface between these subcomplexes. It will be interesting to determine whether this interface is at the hinge region that mediates interaction between Smc5 and Smc6 or, at the head domains of Smc5/6. There are examples of non-SMC subunits binding to either the hinge or head domains of SMC heterodimers. A protein called Cti1 was recently found to associate with the hinge domain of Smc4, a component of condensin (11). Cti1 is homologous to human C1D that associates with genomic DNA and stimulates DNA-dependent protein kinase activity. Cti1 is an essential protein that enhances the repair roles of condensin (11). The head domains of the cohesin heterodimer Smc1/3 are bridged by SCC1/Rad21. The resultant ring structure has the potential to encircle sister chromatids maintaining cohesion (reviewed in references 31 and 40). Proteolytic cleavage of SCC1/Rad21 breaks the cohesin loop allowing segregation of sister chromatids at anaphase. Based on our study and those of others, there is no clear candidate for an SCC1/Rad21 related factor in the Smc5/6 complex, as the essential subunits Nse1 to -4 do not bind Smc6 in our assays (54; data not shown). Smc5/6 may function without enclosing sister chromatids in a proteinaceous loop or perhaps, Nse5/6 may bridge the Smc5/6 head domains to form a repair specific conformation of the complex.
Smc5/6 complexes from yeast to human contain six subunits that are clearly homologous at the primary sequence level (31, 40). Smc5/6 complexes of budding and fission yeasts also contain two additional subunits, KRE29/YML023c and Nse5/6, respectively, which are apparently sequence orphans (20, 66; the present study). It is possible that KRE29/YML023c and Nse5/6 are functionally homologous; however; KRE29 and YML023c are both essential for viability, whereas Nse5 and Nse6 are not. Nse6, and possibly KRE29, are members of the ARM/HEAT repeat family. ARM/HEAT repeats provide protein-protein interfaces and are found in factors associated with both the cohesin and condensin complexes, among other chromatin related proteins (41). Two budding yeast factors that contain ARM/HEAT repeats, SCC2 and PDS5, play central roles in modulating the association of cohesin with chromosomes (31, 40). Although budding and fission yeast PDS5 proteins are required for the stable association of cohesin with chromosomes, PDS5 is only essential for growth in budding yeast. Therefore, that KRE29 is essential but its potential fission yeast counterpart Nse6 is not has precedent among SMC complex components. Clearly, more work is required to test the functional homology of Nse5/6 and KRE29/YML023c.
To define the repair pathways in which Nse5/6 are involved, we have performed extensive genetic analyses. Nse5/6 mutants exhibit spontaneous DNA lesions that are bound by Rad22. These could be DSBs secondary to the breaking of unresolved chromosomes at mitosis or, regions of RPA coated single-stranded DNA that can be generated at stalled replication forks. The Nse5/6 mutants constitutively activate the DNA structure checkpoint defined by Rad3 (hATR) and Chk1. In the absence of the checkpoint, Nse5/6 mutants undergo catastrophic mitoses. When Nse5/6 mutants are UV irradiated and allowed to progress through mitosis, chromosomes are often stretched along the axis of the mitotic spindle. These phenotypes are reminiscent of those described for nse1-1, nse2-1, and nse4-1 (rad62-1) hypomorphic mutants (19, 34, 36).
The particular spectrum of DNA damage sensitivities of Nse5/6 mutants demonstrates a major role for the heterodimer in mitigating replication-associated damage. Nse5/6 mutants are hypersensitive to agents that can stall replication forks (UV, HU, and MMS) as opposed to collapsing them (camptothecin). Nse5/6 mutants are much less sensitive to camptothecin than rhp51 cells, suggesting that Nse5/6 play a minor role, if any, in recombination-dependent replication restart.
We have examined the role played by Nse5/6 in the repair of UV-induced DNA damage in greater detail. Nse5/6 do not solely facilitate the excision repair of UV lesions, which in fission yeast is performed by NER and Uve1 (10, 65). In the absence of UV damage excision repair pathways, deletion of Nse5/6 greatly sensitizes cells to UV. This suggests that Nse5/6 are required for the tolerance of unrepaired UV damage during replication. Tolerance of DNA damage during replication depends heavily on both translesion polymerases (28, 48) and homologous recombination-dependent processes (8, 21, 38).
A striking finding is that the low-dose UV sensitivity of cells lacking Nse6 is suppressed by deleting the HRR factor Rhp51. The same is true for a hypomorphic allele of the essential Nse2 subunit (nse2-1). We can envision two likely explanations for the observed suppression. First, Nse5/6 may normally function downstream of Rhp51 in response to UV damage. In this scenario, the absence of Nse5/6 results in lethality due to a defect in processing the recombination intermediate established by Rhp51. Second, Nse5/6 might normally act to limit or antagonize HRR repair of UV lesions at stalled replication forks. The absence of Nse5/6 might lead to hyper-recombination that could overload the cells capacity to resolve sister chromatids or result in the formation of pathological structures. We were unable to detect elevated spontaneous recombination rates between ade6 heteroalleles in an Nse6 mutant (43; data not shown). This suggests that either Nse5/6 mutation does not result in hyper-recombination or the elevated recombination processes may be lethal in the absence of Nse6.
The mediator complexes Rhp55/57 and Swi5/Sfr1 have both been shown to be required for the function of Rhp51 in response to UV-induced and other DNA damage (2). Therefore, because Rhp51 deletion rescues the low dose UV sensitivity of an Nse6 mutant, we expected that deleting both Rhp55 and Swi5 together would also rescue the Nse6 mutant. Surprisingly, we found that the low-dose UV sensitivity of an nse6 swi5 rhp55 triple mutant (only slightly less sensitive than nse6 alone) was not equivalent to that of the swi5 rhp55 double mutant. This contrasts with the nse6 rhp51 double mutant, which is equally sensitive to UV as the rhp51 single mutant. Deleting Swi5 alone and to a lesser extent Rhp55, weakly rescued Nse6 mutants. These observations suggest that in the absence of Nse5/6, Rhp51 is able to act partially independently of the previously described mediator complexes. It should be noted that deletion of Rhp55 in an nse6 background, in contrast to the deletion of Rhp51 or Swi5, results in a synthetic growth defect. This may reflect additional functions of Rhp55 other than in Rhp51 nucleoprotein filament formation, such as the postsynaptic role recently suggested for Rhp55/57 (22). Such dual roles clearly complicate the epistasis analysis between nse6 and Rhp55.
We have found that Nse5/6 mutants are dependent on Mus81 and Rqh1 for viability. Both Rqh1 and Mus81 have been implicated in the processing of Holliday junctions that can form as a result of HRR (7, 8, 12, 13). Several of the DNA damage sensitivities and repair defects exhibited by Mus81 and Rqh1 mutants can be suppressed by overexpressing the bacterial Holliday junction resolvase RusA (7, 12, 13). Likewise, we find that the UV sensitivity of Nse6 mutants is partially suppressed by RusA overexpression. Further, we observed that the growth of Nse6 mutants was improved even in the absence of exogenous DNA damage. This suggests that, in the absence of Nse5/6, Holliday junctions can accumulate. This accumulation of Holliday junctions may overwhelm the cells ability to resolve them in the absence of either Mus81 or Rqh1. It should be noted that Mus81 might act on recombination intermediates that precede full Holliday junctions, rather than resolving them once formed.
In conclusion, we have identified a novel heterodimer involved in DNA repair. Nse5/6 associates with Smc5/6 and likely influences the repair-specific functions of the holocomplex. We have reconstituted the octameric Smc5/6 holocomplex and have begun to define its architecture. Genetic data suggest that Nse5/6 are important to prevent the accumulation of Holliday junctions when replication forks encounter blocking lesions. Unresolved Holliday junctions may explain the sister chromatid segregation defects prevalent in Nse5/6 and Smc5/6 mutants after UV irradiation.
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ACKNOWLEDGMENTS |
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S.P. is supported by a fellowship from the Swiss Cancer League-KLS-01525-02-2004. J.W. is supported by an American Cancer Society Postdoctoral Fellowship, and J.R.Y. is supported by an NIH grant RR11823-08. This study was funded by NIH grant GM068608 awarded to M.N.B.
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FOOTNOTES |
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M.N.B. dedicates this study to his son Conor.
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, background bands.
