Novel essential DNA repair proteins Nse1 and Nse2 are subunits of the fission yeast Smc5-Smc6 complex

The structural maintenance of chromosomes (SMC) family of proteins play essential roles in genomic stability. SMC heterodimers are required for sister-chromatid cohesion (Cohesin: Smc1 & Smc3), chromatin condensation (Condensin: Smc2 & Smc4), and DNA repair (Smc5 & Smc6). The SMC heterodimers do not function alone and must associate with essential non-SMC subunits. To gain further insight into the essential and DNA repair roles of the Smc5-6 complex, we have purified fission yeast Smc5 and identified by mass spectrometry the co-precipitating proteins, Nse1 and Nse2. We show that both Nse1 and Nse2 interact with Smc5 in vivo, as part of the Smc5-6 complex. Nse1 and Nse2 are essential proteins and conserved from yeast to man. Loss of Nse1 and Nse2 function leads to strikingly similar terminal phenotypes to those observed for Smc5-6 inactivation. In addition, cells expressing hypomorphic alleles of Nse1 and Nse2 are, like Smc5-6 mutants, hypersensitive to DNA damage. Epistasis analysis suggests that like Smc5-6, Nse1, and Nse2 function together with Rhp51 in the homologous recombination repair of DNA double strand breaks. The results of this study strongly suggest that Nse1 and Nse2 are novel non-SMC subunits of the fission yeast Smc5-6 DNA repair complex.     

Nse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex

The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein ‘holo-complex’ is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.     

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo‐complex. We found that the Nse5/6 sub‐complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross‐linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross‐linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non‐productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.     

During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks

The Smc5/6 complex belongs to the SMC (structural maintenance of chromosomes) family, which also includes cohesin and condensin. In Saccharomyces cerevisiae, the Smc5/6 complex contains six essential non-Smc elements, Nse1-6. Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 small ubiquitin-like modifier (SUMO) ligase important for Smc5 sumoylation. Characterization of two temperature-sensitive mutants, nse5-ts1 and nse5-ts2, demonstrated the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, formation of fork-associated homologous recombination intermediates, and hydroxyurea sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, and for its functionality during replication stress.     

Composition and architecture of the Schizosaccharomyces pombe Rad18 (Smc5-6) complex

The rad18 gene of Schizosaccharomyces pombe is an essential gene that is involved in several different DNA repair processes. Rad18 (Smc6) is a member of the structural maintenance of chromosomes (SMC) family and, together with its SMC partner Spr18 (Smc5), forms the core of a high-molecular-weight complex. We show here that both S. pombe and human Smc5 and -6 interact through their hinge domains and that four independent temperature-sensitive mutants of Rad18 (Smc6) are all mutated at the same glycine residue in the hinge region. This mutation abolishes the interactions between the hinge regions of Rad18 (Smc6) and Spr18 (Smc5), as does mutation of a conserved glycine in the hinge region of Spr18 (Smc5). We purified the Smc5-6 complex from S. pombe and identified four non-SMC components, Nse1, Nse2, Nse3, and Rad62. Nse3 is a novel protein which is related to the mammalian MAGE protein family, many members of which are specifically expressed in cancer tissue. In initial steps to understand the architecture of the complex, we identified two subcomplexes containing Rad18-Spr18-Nse2 and Nse1-Nse3-Rad62. The subcomplexes are probably bridged by a weaker interaction between Nse2 and Nse3.     

Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis

The structural maintenance of chromosomes (SMC) family of proteins play key roles in the organization, packaging, and repair of chromosomes. Cohesin (Smc1+3) holds replicated sister chromatids together until mitosis, condensin (Smc2+4) acts in chromosome condensation, and Smc5+6 performs currently enigmatic roles in DNA repair and chromatin structure. The SMC heterodimers must associate with non-SMC subunits to perform their functions. Using both biochemical and genetic methods, we have isolated a novel subunit of the Smc5+6 complex, Nse3. Nse3 is an essential nuclear protein that is required for normal mitotic chromosome segregation and cellular resistance to a number of genotoxic agents. Epistasis with Rhp51 (Rad51) suggests that like Smc5+6, Nse3 functions in the homologous recombination based repair of DNA damage. We previously identified two non-SMC subunits of Smc5+6 called Nse1 and Nse2. Analysis of nse1-1, nse2-1, and nse3-1 mutants demonstrates that they are crucial for meiosis. The Nse1 mutant displays meiotic DNA segregation and homologous recombination defects. Spore viability is reduced by nse2-1 and nse3-1, without affecting interhomolog recombination. Finally, genetic interactions shared by the nse mutants suggest that the Smc5+6 complex is important for replication fork stability.     

Rad62 protein functionally and physically associates with the smc5/smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast

Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination.     

The Smc5-Smc6 DNA repair complex. bridging of the Smc5-Smc6 heads by the KLEISIN, Nse4, and non-Kleisin subunits

Structural maintenance of chromosomes (SMC) proteins play fundamental roles in many aspects of chromosome organization and dynamics. The SMC complexes form unique structures with long coiled-coil arms folded at a hinge domain, so that the globular N- and C-terminal domains are brought together to form a "head." Within the Smc5-Smc6 complex, we previously identified two subcomplexes containing Smc6-Smc5-Nse2 and Nse1-Nse3-Nse4. A third subcomplex containing Nse5 and -6 has also been identified recently. We present evidence that Nse4 is the kleisin component of the complex, which bridges the heads of Smc5 and -6. The C-terminal part of Nse4 interacts with the head domain of Smc5, and structural predictions for Nse4 proteins suggest similar motifs that are shared within the kleisin family. Specific mutations within a predicted winged helix motif of Nse4 destroy the interaction with Smc5. We propose that Nse4 and its orthologs form the delta-kleisin subfamily. We further show that Nse3, as well as Nse5 and Nse6, also bridge the heads of Smc5 and -6. The Nse1-Nse3-Nse4 and Nse5-Nse6 subcomplexes bind to the Smc5-Smc6 heads domain at different sites.     

Nse1 RING-like domain supports functions of the Smc5-Smc6 holocomplex in genome stability

The Smc5-Smc6 holocomplex plays essential but largely enigmatic roles in chromosome segregation, and facilitates DNA repair. The Smc5-Smc6 complex contains six conserved non-SMC subunits. One of these, Nse1, contains a RING-like motif that often confers ubiquitin E3 ligase activity. We have functionally characterized the Nse1 RING-like motif, to determine its contribution to the chromosome segregation and DNA repair roles of Smc5-Smc6. Strikingly, whereas a full deletion of nse1 is lethal, the Nse1 RING-like motif is not essential for cellular viability. However, Nse1 RING mutant cells are hypersensitive to a broad spectrum of genotoxic stresses, indicating that the Nse1 RING motif promotes DNA repair functions of Smc5-Smc6. We tested the ability of both human and yeast Nse1 to mediate ubiquitin E3 ligase activity in vitro and found no detectable activity associated with full-length Nse1 or the isolated RING domains. Interestingly, however, the Nse1 RING-like domain is required for normal Nse1-Nse3-Nse4 trimer formation in vitro and for damage-induced recruitment of Nse4 and Smc5 to subnuclear foci in vivo. Thus, we propose that the Nse1 RING-like motif is a protein–protein interaction domain required for Smc5-Smc6 holocomplex integrity and recruitment to, or retention at, DNA lesions.     

Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage

The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M(r) complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62. The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint. We have characterized Nse2 and show here that it is a SUMO ligase. Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein. Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation. nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal. Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain. Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex.     

Nse1 and Nse4, subunits of the Smc5–Smc6 complex,are involved in Dictyostelium development upon starvation

The Smc5–Smc6 complex contains a heterodimeric core of two SMC proteins and non‐Smc elements (Nse1–6), and plays an important role in DNA repair. We investigated the functional roles of Nse4 and Nse1 in Dictyostelium discoideum. Nse4 and Nse3 expressed as Flag‐tagged fusion proteins were highly enriched in nuclei, while Nse1 was localized in whole cells. Using yeast two‐hybrid assays, only the interaction between Nse3 and Nse1 was detected among the combinations. However, all of the interactions among these three proteins were recognized by co‐immunoprecipitation assay using cell lysates prepared from the cells expressing green fluorescent protein (GFP)‐ or Flag‐tagged fusion proteins. GFP‐tagged Nse1, which localized in whole cells, was translocated to nuclei when co‐expressed with Flag‐tagged Nse3 or Nse4. RNAi‐mediated Nse1 and Nse4 knockdown cells (Nse1 KD and Nse4 KD cells) were generated and found to be more sensitive to UV‐induced cell death than control cells. Upon starvation, Nse1 and Nse4 KD cells had increases in the number of smaller fruiting bodies that formed on non‐nutrient agar plates or aggregates that formed under submerged culture. We found a reduction in the mRNA level of pdsA, in vegetative and 8 h‐starved Nse4 KD cells, and pdsA knockdown cells displayed effects similar to Nse4 KD cells. Our results suggest that Nse4 and Nse1 are involved in not only the cellular DNA damage response but also cellular development in D. discoideum.     

Structure Basis for Shaping the Nse4 Protein by the Nse1 and Nse3 Dimer within the Smc5/6 Complex

The Smc5/6 complex facilitates chromosome replication and DNA break repair. Within this complex, a subcomplex composed of Nse1, Nse3 and Nse4 is thought to play multiple roles through DNA binding and regulating ATP-dependent activities of the complex. However, how the Nse1-Nse3-Nse4 subcomplex carries out these multiple functions remain unclear. To address this question, we determine the crystal structure of the Xenopus laevis Nse1-Nse3-Nse4 subcomplex at 1.7 Å resolution and examine how it interacts with DNA. Our structural analyses show that the Nse1-Nse3 dimer adopts a closed conformation and forms three interfaces with a segment of Nse4, forcing it into a Z-shaped conformation. The Nse1-Nse3-Nse4 structure provides an explanation for how the lung disease immunodeficiency and chromosome breakage syndrome-causing mutations could dislodge Nse4 from Nse1-Nse3. Our DNA binding and mutational analyses reveal that the N-terminal and the middle region of Nse4 contribute to DNA interaction and cell viability. Integrating our data with previous crosslink mass spectrometry data, we propose potential roles of the Nse1-Nse3-Nse4 complex in binding DNA within the Smc5/6 complex.     

Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells

The structural maintenance of chromosomes (SMC) proteins constitute the core of critical complexes involved in structural organization of chromosomes. In yeast, the Smc5/6 complex is known to mediate repair of DNA breaks and replication of repetitive genomic regions, including ribosomal DNA loci and telomeres. In mammalian cells, which have diverse genome structure and scale from yeast, the Smc5/6 complex has also been implicated in DNA damage response, but its further function in unchallenged conditions remains elusive. In this study, we addressed the behavior and function of Smc5/6 during the cell cycle. Chromatin fractionation, immunofluorescence, and live-cell imaging analyses indicated that Smc5/6 associates with chromatin during interphase but largely dissociates from chromosomes when they condense in mitosis. Depletion of Smc5 and Smc6 resulted in aberrant mitotic chromosome phenotypes that were accompanied by the abnormal distribution of topoisomerase IIα (topo IIα) and condensins and by chromosome segregation errors. Importantly, interphase chromatin structure indicated by the premature chromosome condensation assay suggested that Smc5/6 is required for the on-time progression of DNA replication and subsequent binding of topo IIα on replicated chromatids. These results indicate an essential role of the Smc5/6 complex in processing DNA replication, which becomes indispensable for proper sister chromatid assembly in mitosis.     

SUMO ligase activity of vertebrate Mms21/Nse2 is required for efficient DNA repair but not for Smc5/6 complex stability

Nse2/Mms21 is an E3 SUMO ligase component of the Smc5/6 complex, which plays multiple roles in maintaining genome stability. To study the functions of the vertebrate Nse2 orthologue, we generated Nse2-deficient chicken DT40 cells. Nse2 was dispensable for DT40 cell viability and required for efficient repair of bulky DNA lesions, although Nse2-deficient cells showed normal sensitivity to ionising radiation-induced DNA damage. Homologous recombination activities were reduced in Nse2^−/−/− cells. Nse2 deficiency destabilised Smc5, but not Smc6. In rescue experiments, we found that the SUMO ligase activity of Nse2 was required for an efficient response to MMS- or cis-platin-induced DNA damage, and for homologous recombination, but not for Smc5 stability. Gel filtration analysis indicated that Smc5 and Nse2 remain associated during the cell cycle and after DNA damage and Smc5/Smc6 association is independent of Nse2. Analysis of Nse2^−/−/−Smc5⁻ clones, which were viable although slow-growing, showed no significant increase in DNA damage sensitivity. We propose that Nse2 determines the activity, but not the assembly, of the Smc5/6 complex in vertebrate cells, and this activity requires the Nse2 SUMO ligase function.     

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

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-Nse6 [corrected] (Nse5 and Nse6) [corrected] 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 [corrected] 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.     

The Smc5/Smc6/MAGE Complex Confers Resistance to Caffeine and Genotoxic Stress in Drosophila melanogaster

The SMC5/6 protein complex consists of the Smc5, Smc6 and Non-Smc-Element (Nse) proteins and is important for genome stability in many species. To identify novel components in the DNA repair pathway, we carried out a genetic screen to identify mutations that confer reduced resistance to the genotoxic effects of caffeine, which inhibits the ATM and ATR DNA damage response proteins. This approach identified inactivating mutations in CG5524 and MAGE, homologs of genes encoding Smc6 and Nse3 in yeasts. The fact that Smc5 mutants are also caffeine-sensitive and that Mage physically interacts with Drosophila homologs of Nse proteins suggests that the structure of the Smc5/6 complex is conserved in Drosophila. Although Smc5/6 proteins are required for viability in S. cerevisiae, they are not essential under normal circumstances in Drosophila. However, flies carrying mutations in Smc5, Smc6 and MAGE are hypersensitive to genotoxic agents such as ionizing radiation, camptothecin, hydroxyurea and MMS, consistent with the Smc5/6 complex serving a conserved role in genome stability. We also show that mutant flies are not compromised for pre-mitotic cell cycle checkpoint responses. Rather, caffeine-induced apoptosis in these mutants is exacerbated by inhibition of ATM or ATR checkpoint kinases but suppressed by Rad51 depletion, suggesting a functional interaction involving homologous DNA repair pathways that deserves further scrutiny. Our insights into the SMC5/6 complex provide new challenges for understanding the role of this enigmatic chromatin factor in multi-cellular organisms.     

Interaction mapping between Saccharomyces cerevisiae Smc5 and SUMO E3 ligase Mms21

The multisubunit Smc5-Smc6 holocomplex (Smc5/6) plays a critical role in chromosome stability maintenance, DNA replication, homologous recombination, and double-stranded DNA damage repair. Smc5 and Smc6 form the core of the holocomplex, along with six non-SMC elements, for which most functions are not yet understood. Mms21 (Nse2), the relatively well-studied subunit in Smc5/6, contains a SP-like-RING finger motif on the C-terminus and was identified as a SUMO E3 ligase. Deletion of Mms21 is lethal; however, while deficient in DNA damage repair, SUMO ligase mutants remain viable. These functions of Mms21 in Smc5/6 are hard to address without understanding the interaction between Smc5 and Mms21. Previously, we systematically examined the architecture of Saccharomyces cerevisiae Smc5/6 and, using yeast two-hybrid methods, found that Mms21 interacts with the coiled-coil of Smc5. Later, crystallographic studies revealed the molecular arrangement of Mms21 with Smc5/6. For this study, we use a combination of limited proteolysis, mass spectrometry, and N-terminal sequencing to precisely define the interaction region of Smc5 with Mms21. In addition, using isothermal titration calorimetry, we find that Mms21 interacts with Smc5 in a 1:1 ratio with a K(d) of 0.68 μM. This combination of methods would be useful in examining the structure of any large multiprotein complex.     

Chromatin loading of Smc5/6 is induced by DNA replication but not by DNA double-strand breaks

Smc6, a member of the structural maintenance of chromosomes (SMC) family of proteins, forms a complex with related Smc5. Genetic analyses of yeast have demonstrated the involvement of Smc6 in DNA repair and checkpoint responses. In this study, we investigated the role of the Smc5/6 complex in higher eukaryotes by analyzing its behavior in Xenopus laevis egg extracts. Smc5/6 was loaded onto chromatin during DNA replication in a manner dependent on the initiation of DNA synthesis, and it dissociated from chromatin during mitosis. Moreover, the induction of DNA double-strand breaks following replication did not significantly affect the amount of chromatin-associated Smc6. These findings suggest that the Smc5/6 complex is regulated during the cell cycle, presumably in anticipation of DNA damage that may arise during replication.     

H2A.Z-Dependent Regulation of Cohesin Dynamics on Chromosome Arms

Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation.     

The Nse2/Mms21 SUMO ligase of the Smc5/6 complex in the maintenance of genome stability

There exist three highly-conserved structural maintenance of chromosomes (Smc) complexes that ensure genome stability during eukaryotic cell division. There are the well-characterized cohesin and condensin complexes and the third Smc complex, Smc5/6. Nse2/Mms21, a SUMO ligase, is a component of the Smc5/6 complex and recent data have indicated that Nse1 may function as a ubiquitin ligase. Smc5/6 regulates sister chromatid cohesion, homologous recombination and chromatin structure and conformation. This review examines the functions of Smc5/6 in DNA repair and the maintenance of genomic integrity and explores the roles of the associated SUMO and ubiquitin ligases. Recent findings have indicated that Smc5/6 may play a topological role in chromosome dynamics, which may help understand the complexity of its activities.