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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.

The Nse5-Nse6 Dimer Mediates DNA Repair Roles of the Smc5-Smc6 Complex{dagger}

Stephanie Pebernard,1 James Wohlschlegel,2 W. Hayes McDonald,2 John R. Yates III,2 and Michael N. Boddy1*

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
 
Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nes6 (Nse5/6) mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 mutants is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.


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INTRODUCTION
 
The integrity of the genome is threatened during the intricate processes of replication and mitotic chromosome segregation. Therefore, multiple mechanisms have evolved to ensure the fidelity of chromosome transmission. Critical among these mechanisms are those of DNA repair and DNA packaging. An intriguing family of proteins at the core of the structural maintenance of chromosomes complexes (SMC) play pivotal roles in both mechanisms (31, 40). Heterodimers of SMC1 to -6 form the basis of three fundamental complexes: (i) cohesin that maintains sister chromatid cohesion after replication (SMC1-SMC3 [SMC1/3]), (ii) condensin that is involved in compaction and decatenation of sister chromatids at mitosis (SMC2/4), and (iii) SMC5/6 that has essential but largely undefined roles in chromosome organization (31, 40).

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
 
Strains. All strains are ura4-D18 leu1-32 unless otherwise stated: PR100, h+; PR109, h–; NBY460, h+ smc5:TAP:kanMx6; NBY782, nse4:TAP:kanMx6 nse1:myc:kanMx6; NBY852, nse6:TAP:kanMx6 nse1:myc:kanMx6; NBY853, nse6:TAP:kanMx6 nse2:myc:kanMx6; SP46, nse6:TAP:kanMx6 myc:Smc6:ura4+; NBY900, nse5:TAP:kanMx6 nse1:myc:kanMx6; NBY901, nse5:TAP:kanMx6 nse6:myc:kanMx6; NBY893, h+ nse5:myc:kanMx6; NBY848, h+ nse6:myc:kanMx6; NBY895, h– nse5::kanMx6; NBY835, h+ nse6::kanMx6; NBY863, nse6::kanMx6 chk1::ura4+; NBY880, nse6::kanMx6 rad3::ura4+; NBY491, rad22:YFP:kanMx6; NBY884, Rad22:YFP:kanMx6 nse6::kanMx6; NBY903, nse5::ura4+ nse6::kanMx6; PS2345, rhp51::ura4+; NBY855, nse6::kanMx6 rhp51::ura4+; NBY527, h+ nse2-1:myc:kanMx6; NBY547, nse2-1:myc:kanMx6 rhp51::ura4+; NBY937, h+ rhp55::ura4+ leu1-32 ura4-D18 ade7-152; NBY914, nse6::kanMx6 rhp55::ura4+ leu1-32 ura4-D18 ade7-152; NBY910, h+ swi5::kanMx6; NBY953, nse6::kanMx6 swi5:myc::LEU2+; NBY955, nse6::kanMx6 swi5:myc::LEU2+ rhp55::ura4+ leu1-32 ura4-D18 ade7-152; NBY188, rad13::ura4+; NBY885, nse6::kanMx6 rad13::ura4+; PR2402, uve1::LEU2+; NBY886, nse6::kanMx6 uve1::LEU2+; NBY887, nse6::kanMx6 rad13::ura4+ uve1::LEU2+; NBY523A, h– rhp18::kanMx6; NBY921, h+ nse6::kanMx6 rhp18::kanMx6; NBY899, nse6:myc::ura4+ brc1::kanMx6; NBY934, h– nse6::kanMx6 pSLF173L:LEU2+; NBY935, h+ nse6::kanMx6 pSLF173L:LEU2+; NBY933, h– nse6::kanMx6 pREP1-Brc1:LEU2+; NBY932, h+ nse6::kanMx6 pREP1-Brc1:LEU2+; NBY916, h– nse6::kanMx6 pREP1-NLS-RusA:LEU2+; NBY915, h+ nse6::kanMx6 pREP1-NLS-RusA:LEU2+; NBY936, h– nse6::kanMx6 pREP1-NLS-RusAD70N:LEU2+; and NBY917, h+ nse6::kanMx6 pREP1-NLS-RusAD70N:LEU2+.

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 {Delta}Cn value greater than 0.08.


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RESULTS
 
Nse5 and Nse6, novel components of the fission yeast Smc5/6 complex. We previously identified four essential non-SMC subunits of the fission yeast Smc5/6 complex, Nse1 to -4 (34, 44). Interestingly, the budding yeast complex consists of the SMC5/6 heterodimer plus six essential subunits (20, 66). Four of these, NSE1-4, are homologous to the fission yeast Nse1 to -4 subunits (34, 36, 44). However, sequence homologues of the two remaining subunits, KRE29 and YML023c, are not detectable in fission yeast or other species. Given the otherwise strong compositional conservation of the Smc5/6 complex, we hypothesized that there may in fact be functional homologues of KRE29 and YML023c in fission yeast, which had diverged at the sequence level. To test this hypothesis, we affinity purified epitope-tagged Nse4 (Nse4-TAP) from fission yeast and identified copurifying proteins by mass spectrometry, as previously described for Smc5-TAP (34, 44). As expected, Smc5, Smc6, and Nse1 to -4 were all identified in the Nse4-TAP data set (Table 1). We then compared the Nse4-TAP and Smc5-TAP datasets for novel proteins found in both purifications, but not in datasets of purifications of unrelated proteins. This approach led to the identification of two uncharacterized proteins, SPBC651.10 and SPAC11E3.08c (Table 1). In keeping with the established nomenclature for subunits of the Smc5/6 complex, we propose that these proteins be named Nse5 and Nse6, respectively. We also purified Nse6-TAP from fission yeast and analyzed copurifying proteins by mass spectrometry, revealing good sequence coverage of Smc5/6 and Nse1 to -6 (Table 1).


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  		TABLE 1. Mass spectrometric analysis of proteins copurifying with Smc5/6 complex factorsa

The interaction between Nse6 and the Smc5/6 complex was further confirmed by immunoprecipitation of endogenous proteins. Strains were generated in which Nse1, Nse2, and Smc6 were tagged at their genomic locus with the myc epitope. In these strains, Nse6 was also tagged at its genomic locus with the TAP epitope. Affinity purification of Nse6-TAP resulted in a robust and specific coprecipitation of Nse1-myc, Nse2-myc, and myc-Smc6 (Fig. 1A). As expected, affinity-purified Nse5-TAP also coprecipitates with elements of the Smc5/6 complex, including Nse1-myc and Nse6-myc (Fig. 1B and C). The preceding data strongly suggest that Nse5 and Nse6 represent novel components of the Smc5/6 complex.


Figure 1
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  		FIG. 1. In vivo confirmation of the Nse5/6 interaction with the Smc5/6 complex. Strains expressed the indicated epitope-tagged proteins from their genomic loci, under the control of their endogenous promoters. Lysates were prepared from each strain and incubated with IgG-Sepharose to recover Nse6-TAP. A fraction of each lysate was loaded as a input control (left panel) prior to immunoprecipitation (right panel). Samples were resolved by SDS-PAGE and immunoblotted with antisera to either the TAP or myc epitopes.

Nse5 and Nse6, like KRE29 and YML023c in budding yeast, have no sequence homologues in other species. However, it is noteworthy that Nse6 belongs to the ARM/HEAT repeat-containing family of proteins, as determined by using the 3D-PSSM structure-prediction program (25). Two ARM/HEAT repeat-containing proteins, SCC2 and Pds5, are required for cohesin's stable association with chromatin (reviewed in references 31 and 40). In addition, condensin possesses two ARM/HEAT-repeat containing subunits. Whether or not they act as "loading" factors like SCC2 is not known. The presence of ARM/HEAT-repeat proteins in the three SMC complexes suggests that they may play related roles in regulating the chromatin association of the holocomplexes.

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).


Figure 2
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  		FIG. 2. Analysis of protein-protein interactions within the Smc5/6 complex expressed in insect cells. (A) Control for background binding to anti-Flag affinity resin. Insect cells were simultaneously infected with baculoviruses to express the indicated epitope-tagged Smc5/6 complex components. After lysis, an input control sample was taken (lane I), and the rest was incubated with anti-Flag beads (lane F). Samples were resolved by SDS-PAGE and immunoblotted with antisera to the indicated epitopes. (B) Same as for panel A but note that the experiment depicted in lanes 1 and 2 did not include Smc6 and Flag-Smc5 was precipitated. (C and D) Same as for panel A, except Flag-Nse5 and Flag-Smc5 were precipitated, respectively. The proteins in each of the lanes I (input) and F (IP with anti-Flag) of part D are as follows: lanes 1 and 2, Flag-Smc5; lanes 3 and 4, Flag-Smc5 and HA-Nse1; lanes 5 and 6, Flag-Smc5 and HA-Nse3; lanes 7 and 8, Flag-Smc5 and HA-Nse4; lanes 9 and 10, Flag-Smc5, HA-Nse3 and HA-Nse1; lanes 11 and 12, Flag-Smc5, HA-Nse1, HA-Nse3 and HA-Nse4; lanes 13 and 14, Flag-Smc5 and HA-Nse2; and lanes 15 and 16, Flag-Smc5, HA-Nse1, HA-Nse2, HA-Nse3, and HA-Nse4. (E) No interaction between Nse1 to -4 and Smc6 is detected in the absence of Smc5. Insect cells expressing the indicated proteins were lysed and His-Smc6 was purified on a nickel-nitrilotriacetic acid column. Proteins copurifying with His-Smc6 were detected by anti-HA immunoblotting. Inputs (lanes I) and His-Smc6 pull-downs are shown (lanes H). Figure 2, background bands.

To analyze the place held by Nse5 and Nse6 in the complex, we coexpressed and immunoprecipitated each of these two proteins in combination with all of the other Smc5/6 complex subunits. Strikingly, we found that the octameric Smc5/6 holocomplex could be reconstituted in insect cells. When FLAG-Smc5 was purified from insect cells simultaneously expressing all eight subunits, a robust coprecipitation of Smc6 and Nse1 to -6 was observed (Fig. 2B, lanes 3 and 4). To our knowledge, this is the first time the complex has been reconstituted in a heterologous system. Stable subcomplexes of Nse1/3/4 and Smc5/Nse2 were recently identified, but no robust interaction between these subcomplexes was observed (54). Notably, the interaction between Smc5 and the other components of the complex does not depend on the presence of Smc6 (Fig. 2B, lanes 1 and 2). Indeed, no interaction between Nse1 to -4 and Smc6 was observed in the absence of Smc5 (Fig. 2E). This observation suggests that Smc5 is at the core of the complex, acting as a "platform" for interaction with Nse1 to -6 and Smc6. One possible explanation for the difference between our results and those of Sergeant et al. is the presence of the additional Nse5/6 components in the complex (54). We believe that bridging of the Nse1/3/4 and Smc5/Nse2 subcomplexes by Nse5/6 is unlikely, since there is no detectable interaction between Nse5/6 and the Nse1 to -4 subunits (Fig. 2C). Alternatively, the differences may arise from protein folding and posttranslational modification issues in the system used in the present study versus those previously used (54). These data do, however, demonstrate a robust interaction between Nse5 and Nse6 (Fig. 2C, lane 2).

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).


Figure 3
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  		FIG. 3. Nse5/6 bind the Smc5/6 heterodimer through multiple contacts. (A) GST-Smc6 was expressed in insect cells together with HA-Nse6, lanes 1 and 2; FLAG-Nse5, lanes 3 and 4; or both, lanes 5 and 6. Lysates were made, and input samples (lanes I) and samples after GSH-Sepharose pull-down of GST-Smc6 (lanes G) were resolved by SDS-PAGE, followed by immunoblotting with anti-HA/FLAG antisera. HA-Nse6, FLAG-Nse5, HA-Smc5, His-Smc6, or epitope-tagged Nse1 to -4 showed no affinity for the GSH-Sepharose resin or GST itself (data not shown). (B) Same as for panel A except that HA-Smc5 was included in all experiments and lanes 1 and 2 were not infected with Nse5/6. Figure 3, position of a background band visible just below the position of HA-Nse6 in all GST pull-down lanes. (C) Confirmation of interactions above by release of soluble complexes from GSH-Sepharose columns using the PreScission protease. GST-Nse5, containing a PreScission site between the GST and Nse5 domains, was used to coinfect insect cells with HA-Nse6 (lane 1), HA-Smc5 (lane 2) or His-Smc6 (lane 3). After recovery of GST-Nse5 from lysates on GSH-Sepharose, Nse5 and associated proteins were specifically eluted by using the PreScission protease. (D) Same as for panel C but GST-Nse6 was used instead. (E) Reconstitution and PreScission protease-dependent elution of a soluble octameric Smc5/6 holocomplex. GST-Smc5 (lane 1) or GST-Smc6 (lane 2) containing the PreScission protease site was used to coinfect insect cells together with the other seven subunits of the complex, epitope-tagged as indicated. After recovery of the GST fusion proteins on GSH-Sepharose, the soluble octameric Smc5/6 complex was eluted by addition of PreScission protease. Proteins were detected by immunoblotting with the indicated antisera or by amido black staining of the membrane (Smc5/6). (F) Revised model of the architecture of the octameric Smc5/6 holocomplex. Key changes are the robust interaction observed between the Nse1/3/4 trimer and Smc5 and the addition of the Nse5/6 heterodimer that appears to make multiple contacts with both Smc5 and Smc6.

To further confirm these interactions, we performed reciprocal precipitations with GST-Nse5 or GST-Nse6. In these experiments, GST-Nse5 or GST-Nse6 was expressed in insect cells, in the presence of Nse6 or Nse5, respectively, or Smc5 or Smc6. The GST-Nse5/6 fusion proteins contain a PreScission protease site, allowing specific protease-dependent release of Nse5/6 from the GSH-Sepharose column. When GST-Nse5 was coexpressed with Nse6, precipitated, and released from the column, a relatively stoichiometric soluble complex of Nse5/6 was recovered (as judged by Coomassie blue staining [Fig. 3C]). Both Smc5 and Smc6 showed affinity for Nse5, which was independent of Nse6 (Fig. 3C). The same pattern of affinities was observed in the reciprocal experiment using GST-Nse6 (Fig. 3D). These data confirm that Nse5/6 bind the Smc5/6 heterodimer, apparently via multiple contacts between the different subunits. Finally, we found that the octameric Smc5/6 holocomplex could be precipitated from insect cell extracts via GST-Smc5 or GST-Smc6 and released in a soluble form by addition of the PreScission protease (Fig. 3E). The aforementioned studies provide detailed information about the direct contacts made between components of the Smc5/6 holocomplex; however; they do not provide information on the stoichiometry of the subunits. Based on the interaction data presented, we propose a modified architectural model for the Smc5/6 holocomplex (Fig. 3F). Nse1/3/4 and Nse2 bind independently to Smc5. The Nse5/6 dimer binds to the Smc5/6 dimer independently of Nse1 to -4. Where Nse1 and Nse3 to -6 contact the Smc5/6 dimer is currently unknown, whereas Nse2 is thought to bind the coiled-coil region proximal to the Smc5 WalkerA/B motifs (54).

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).


Figure 4
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  		FIG. 4. Nse5/6 are novel nuclear factors that prevent spontaneous DNA damage and facilitate repair/segregation of DNA. (A) Nse5 and Nse6, myc tagged at their genomic loci, were detected in cells by indirect immunofluorescence with a monoclonal anti-myc antibody. (B) The DNA structure checkpoints are constitutively active in Nse6 mutants, and chromosomes fail to segregate properly after DNA damage. In the top two panels, wild-type (Wt) cells are shown without treatment (left) and after UV irradiation at 100 J/m2 (right). Nuclei stained with DAPI (4',6'-diamidino-2-phenylindole) have been merged with the corresponding phase images to more clearly show cell cycle stage and mitotic abnormalities. Nse6 mutant cells (nse6{Delta}) are shown below the wild type. Arrowheads indicate aberrantly long cells (left) or mitotic abnormalities after UV irradiation (right). The last two panels show double mutants of nse6{Delta} with enforcers of the DNA structure checkpoints, Chk1 and Rad3, in the absence of extrinsic DNA damaging agents. Arrowheads indicate aberrant mitoses, including aneuploid or "cut" cells. (C) Nse6 mutants exhibit high levels of spontaneous DNA damage, as judged by the formation of foci of the HRR factor Rad22. Quantification of the percentage of wild-type or nse6{Delta} cells containing at least one Rad22-YFP focus, in the absence of extrinsic DNA-damaging agents.

We also replaced the entire open reading frames of nse5 and nse6 with the kanMx6 drug resistance cassette, using the previously described one-step PCR procedure (5). Haploid cells lacking either gene are viable but display heterogeneity in cell length (Fig. 4B). This phenotype suggests that Nse5 and Nse6 deleted cells may be undergoing spontaneous DNA damage, which results in the activation of the DNA structure checkpoints (50). Consistently, nse6{Delta} 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{Delta} mutation is very sick when combined with a deletion of the master checkpoint regulator, Rad3 (hATR) (50). The nse6{Delta} rad3{Delta} 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{Delta} cells (Fig. 4C). This suggests that, consistent with the observed DNA damage checkpoint activation, nse6{Delta} 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.


Figure 5
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  		FIG. 5. Nse5/6 form a functionally interdependent heterodimer required for DNA repair. (A) Serial dilutions of the indicated strains were spotted onto media selective for the indicated plasmids, pE277 (vector control) and pE277-nse6 (genomic nse6). One set of plates contained 5 mM HU. The other plates were left untreated (no treatment) or were irradiated with UV at 50 J/m2. Plates were grown at 32°C for 3 to 5 days. (B) Nse5 and Nse6 mutants are epistatic in the UV DNA damage response. Wild-type, single nse5 or nse6 mutants, and the nse5 nse6 double mutant were serially diluted onto YES plates. Plates were left unirradiated or were treated with UV irradiation at 20 J/m2. This dose strongly inhibits but does not prohibit growth of the mutants, allowing comparison of sensitivity. (C) Nse6 mutant cells are hypersensitive to lesions that block replication forks and mildly sensitive to replication fork collapsing agents. The indicated strains were serially diluted and plated on YES plates containing MMS (0.005%) or camptothecin (1 µM). Plates were incubated at 32°C for 3 to 5 days. (D) The indicated strains were diluted and plated on YES plates and then irradiated with 0, 25, 50, 75, and 100 Gy of gamma irradiation. Plates were incubated at 32°C for 3 to 5 days.

Nse6 mutant cells are also hypersensitive to the alkylating agent MMS but only mildly sensitive to the topoisomerase 1 inhibitor camptothecin (Fig. 5C). MMS causes base modifications that can block replicative polymerases, whereas camptothecin causes replication fork collapse, producing a one-sided DSB (45, 46). This one-sided DSB must be "recaptured" in a homologous recombination-dependent process that restarts replication. The extreme sensitivity of HRR mutants such as rhp51{Delta} 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{Delta} rhp51{Delta} double mutants are no more sensitive to gamma irradiation than rhp51{Delta} 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{Delta} and nse2-1 or any other hypomorphic component of the Smc5/6 complex (Table 2). Interestingly, deletion of Rhp51 rescues the sensitivity of nse6{Delta} and nse2-1 cells to UV at low UV doses (Fig. 6A). This observation suggests that in nse6{Delta} 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).


Figure 6
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  		FIG. 6. Deletion of the HRR factor Rhp51 suppresses the sensitivity of Nse6 and Nse2 mutants to low doses of UV. (A) Serial dilutions of the indicated strains were plated on YES plates and subjected to 20 or 50 J of UV irradiation/m2 (left panel). The right panel shows a graphical representation of the spot assay data. The indicated strains were diluted and plated at 800 cells/plate, followed by irradiation with the indicated doses of UV. Plates were incubated at 25°C (permissive temperature of nse2-1) for 5 to 7 days before counting. (B) Deletion of Swi5 provides a slight rescue of the Nse6 mutant compared to that obtained with Rhp51 deletion. The indicated strains were serially diluted onto YES plates, irradiated with the indicated UV dose and incubated at 32°C for 3 to 5 days. (C) Unlike deletion of Rhp51, concomitant deletion of Rhp55 and Swi5 in an nse6 mutant does not lead to a robust rescue of nse6 UV sensitivity. The indicated strains were serially diluted onto YES plates, irradiated with the indicated UV dose, and incubated at 32°C for 3 to 5 days.


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  		TABLE 2. Summary of genetic interactions observed with the nse6{Delta} mutanta

To further define the genetic requirements for the formation of the UV-induced pathological structures in Nse6 mutants, we studied the role of the currently known Rhp51 accessory factors, the Swi5/Sfr1 and Rhp55/Rhp57 heterodimers (2). It has previously been shown that cells lacking Rhp51 are as sensitive to high UV doses as cells that lack both Swi5 and Rhp57 (2). This observation suggests that the repair functions of Rhp51 require at least one of the accessory complexes, Swi5/Sfr1 or Rhp55/Rhp57. Since the low-dose UV sensitivity of Nse6 mutants depends on Rhp51, we tested whether deletion of Rhp55 and/or Swi5 could suppress the UV sensitivity of Nse6-null cells. Deletion of Swi5 and to a lesser extent Rhp55, weakly rescued the UV sensitivity of the Nse6 mutant (Fig. 6B and C). Unexpectedly, based on the relatively robust rescue of nse6 by rhp51, concomitant deletion of Swi5 and Rhp55 in the Nse6 mutant failed to rescue its UV sensitivity more robustly than either single mutant (rhp55 or swi5; Fig. 6C). Therefore, in the Nse6 mutant background, the UV-induced toxic structures formed by Rhp51 are only partially dependent on the mediators Swi5 and Rhp55.

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{Delta} and various representatives of the pathways described above. We found that nse6{Delta} cells are more sensitive to UV than either rad13{Delta} or uve1{Delta} single mutants (Fig. 7A). The nse6{Delta} rad13{Delta} and nse6{Delta} uve1{Delta} 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{Delta} rad13{Delta} uve1{Delta} triple mutant is profoundly more sensitive to UV than the rad13{Delta} uve1{Delta} 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{Delta} 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.


Figure 7
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  		FIG. 7. Nse5/6 function in a DNA damage tolerance pathway. (A) Loss of Nse6 function is additive with defects in NER and the alternate UV repair pathway initiated by Uve1. The indicated strains were serially diluted on YES plates and left untreated or else irradiated with UV irradiation at 10 J/m2 and incubated at 32°C for 3 to 5 days. (B) Nse6 is required for the bypass of irreparable UV lesions during replication. The methods were as described for panel A above. (C) Nse6 has functions that are independent of the post-replicative repair regulator Rhp18 (RAD18). Methods are as described for panels A and B above.

Genetic interactions of Nse5/6 suggest that they stabilize and/or repair stalled replication forks. We previously reported that hypomorphic mutants of the Smc5/6 complex are dependent on the Mus81-Eme1 endonuclease and the Rqh1 DNA helicase for viability (44). These protein factors are strongly implicated in replication fork processing and stabilization (1, 24). Like Smc5/6 mutants, we find that Nse6 mutants are dependent on Mus81-Eme1 and Rqh1 for viability (Fig. 8A). We were unable to retrieve double mutants by either random spore analysis or tetrad dissection. On tetrad dissection plates, presumptive mus81 nse6 double mutants formed microcolonies of approximately 30 cells before growth ceased, whereas rqh1 nse6 double mutants did not proceed beyond two to six cells. We also found that nse6 rad60 double mutants are not viable (Fig. 8A). Rad60 is a recombination repair factor that was identified through its interaction with the replication checkpoint kinase Cds1, the Smc5/6 complex and, through a rad2 synthetic lethal screen (9, 37). Based on physical and genetic interaction data, Rad60 likely plays roles intimately associated with those of the Smc5/6 complex in chromosome organization and repair (9, 37). The genetic interactions described above for the nse6{Delta} 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.


Figure 8
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  		FIG. 8. Nse5/6 mitigate replication-associated damage and are implicated in Holliday junction processing/prevention. (A) The viability of Nse6 mutants is dependent on the activity of protein factors that play critical roles in replication fork protection. Nse6 mutants were crossed with Rqh1, Mus81, and Rad60 mutants, and the meiotic products were tested by random spore and tetrad analysis. The arrows indicate synthetic lethal interactions. (B) Overexpression of the bacterial resolvase RusA partially rescues the UV sensitivity of Nse6 mutants. The Nse6 mutant strain (both mating types h+/h–), transformed with the indicated plasmids, was grown in liquid media that was selective for the plasmid and also allowed for overexpression of each protein from the potent nmt1 promoter. The strains were then serially diluted and plated on solid selective media that maintained protein expression. The plates were left untreated or were irradiated with the indicated doses of UV irradiation and grown for 3 to 5 days at 32°C.

The bacterial resolvase RusA partially suppresses the UV sensitivity of the Nse6 mutant. Ectopic expression of the bacterial resolvase RusA can suppress certain phenotypes of yeast cells deleted for proteins involved in the processing of Holliday junctions, or their predecessors. The RecQ family of DNA helicases process recombination structures, including those at stalled replication forks, apparently resolving and/or preventing the formation of Holliday junctions (26). Fission yeast cells deleted for a RecQ family member, Rqh1, display a number of hypersensitivities to genotoxic agents. These sensitivities are partially suppressed by overexpression of RusA (13). Further, a number of phenotypes resulting from deletion of the fission yeast structure-specific endonuclease Mus81-Eme1 can be fully or partially suppressed by RusA overexpression (7, 12, 55). Mus81-Eme1 has been suggested to cleave multiple recombination intermediates, ranging from 3' flap structures to intact Holliday junctions (7, 12, 55).

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{Delta} 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
 
We have described the identification of new components of the Smc5/6 complex in fission yeast. The discovery of the functionally interdependent heterodimer Nse5/6 has important ramifications. First, the finding that both of the Smc5/6 complexes in the distantly related yeasts are octameric suggests that Nse5/6 functional homologues await discovery in other species (66; the present study). Second, Nse5/6 are nonessential, but mutants display many (not all) of the DNA repair phenotypes observed for hypomorphic mutants of Smc5/6. Thus, the Nse5/6 dimer specifically facilitates some of the DNA repair functions of the Smc5/6 complex.

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{Delta} 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{Delta} swi5{Delta} rhp55{Delta} triple mutant (only slightly less sensitive than nse6 alone) was not equivalent to that of the swi5{Delta} rhp55{Delta} double mutant. This contrasts with the nse6{Delta} rhp51{Delta} 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
 
We thank Clare McGowan and Curt Wittenberg for critical reading of the manuscript. We also thank members of the Scripps Cell Cycle Groups for support and encouragement.

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
 
* Corresponding author. Mailing address: The Scripps Research Institute, Rm. MB107, 10550 North Torrey Pines Rd., Molecular Biology, MB-3, La Jolla, CA 92037. Phone: (858) 784-7042. Fax: (858) 784-2265. E-mail: nboddy{at}scripps.edu. Back

{dagger} M.N.B. dedicates this study to his son Conor. Back


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Molecular and Cellular Biology, March 2006, p. 1617-1630, Vol. 26, No. 5
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