The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex.

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).