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.     

Smc5-Smc6-Dependent Removal of Cohesin from Mitotic Chromosomes

The function of the essential cohesin-related Smc5-Smc6 complex has remained elusive, though hypomorphic mutants have defects late in recombination, in checkpoint maintenance, and in chromosome segregation. Recombination and checkpoints are not essential for viability, and Smc5-Smc6-null mutants die in lethal mitoses. This suggests that the chromosome segregation defects may be the source of lethality in irradiated Smc5-Smc6 hypomorphs. We show that in smc6 mutants, following DNA damage in interphase, chromosome arm segregation fails due to an aberrant persistence of cohesin, which is normally removed by the Separase-independent pathway. This postanaphase persistence of cohesin is not dependent on DNA damage, since the synthetic lethality of smc6 hypomorphs with a topoisomerase II mutant, defective in mitotic chromosome structure, is also due to the retention of cohesin on undamaged chromosome arms. In both cases, Separase overexpression bypasses the defect and restores cell viability, showing that defective cohesin removal is a major determinant of the mitotic lethality of Smc5-Smc6 mutants.Three essential SMC (structural maintenance of chromosomes) complexes control chromosome dynamics: condensin, cohesin, and the Smc5-Smc6 complex (37). They are composed of SMC heterodimers: Smc2 and -4 in condensin, Smc1 and -3 in cohesin, and Smc5 and -6 in Smc5-Smc6. These are large ATPases with globular N and C termini, which are separated by long coiled-coil domains. The termini interact through an ABC-like coordination of ATP through Walker A and B motifs, with the coiled-coils bending at a flexible “hinge” that acts as the SMC dimerization domain. Each complex contains a number of unique non-Smc subunits, which are likely to contribute to its unique function. Among these is a kleisin subunit, which interacts with both the SMC subunits, closing a potential ring-shaped structure (55, 61).Condensin is localized to chromosomes primarily during mitosis and is essential for mitotic chromosome condensation. Conversely, cohesin is localized primarily to interphase chromosomes and has been postulated to form a ring-shaped structure around sister chromatids to ensure their cohesion, which is important for DNA repair by homologous recombination (HR). As its name suggests, the function of the Smc5-Smc6 complex is relatively poorly understood.Scc2/4 loads cohesin onto chromosomes in G₁, and sister chromatid cohesion is established during replication via the action of the acetyltransferase Eco1. Cohesin must be removed before chromosome segregation, where cleavage of the kleisin subunit Scc1 by the protease Separase is critical (51). In Saccharomyces cerevisiae, Separase-mediated Scc1 cleavage is essential for the removal of cohesin from all loci. In mammals, most cohesin is removed from chromosome arms early in mitosis in a Separase-independent process regulated by cohesin phosphorylation (28, 76). At anaphase, Separase-dependent removal of cohesin at the kinetochores ensures sister chromatid separation. In Schizosaccharomyces pombe, cohesin is thought to be regulated in a manner similar to that in mammals; only a small fraction of the Scc1 homolog Rad21 is cleaved by Separase (70), suggesting that most cohesin is removed by a Separase-independent mechanism.Cohesin-mediated sister chromatid cohesion is required for HR (64). Cohesin is recruited to double-stranded DNA breaks (DSBs) (66) and enforces cohesion genome wide after DNA damage in S. cerevisiae (65, 74). The acetyltransferase activity of Eco1 is essential for genomewide damage-induced cohesion, acting via the acetylation of Smc3 (6, 73, 81). In human cells, small interfering RNA (siRNA) studies have suggested a requirement for Smc5-Smc6 to recruit cohesin to DSBs (57), but this is not the case in S. cerevisiae (65), so the functional relationship between these related complexes also remains to be determined.In S. cerevisiae, Smc5-Smc6 is loaded onto chromatin by the cohesin loader Scc2/4 at loci that overlap with cohesin, including at DSBs (36). Smc5-Smc6-null mutants of S. pombe die in aberrant mitoses (27, 75), though the cause of this is unknown. Genetic analyses of Smc5-Smc6 in these yeasts have focused on its role in DNA repair by utilizing viable hypomorphic mutants that are highly sensitive to DNA damage. Studies with two hypomorphic smc6 mutants, bearing the smc6-X and smc6-74 mutations, have shown that Smc5-Smc6 is required for a late stage of HR subsequent to the recruitment of the Rad51/Rad52 recombination proteins and the formation of recombination intermediates (2). smc6-74 is a mutation (A151T) in the arginine finger motif of the N-terminal globular domain, while smc6-X is a mutation (R706C) in the hinge domain. Overexpression of Brc1, a multi-BRCT domain protein, suppresses the DNA damage sensitivities of several Smc5-Smc6 mutants but does not suppress smc6-X (45, 75). smc6-74 mutants, but not smc6-X mutants, are also defective in an early response to replication fork stalling, involving the recruitment of Rad52 but not Rad51 (30).As with cohesin, the HR defects in Smc5-Smc6 hypomorphic mutants are likely to result from a more general role in chromosome organization than acting as a recombinase. Smc5-Smc6 is required for HR following irradiation or recovery from hydroxyurea (HU)-induced replication arrest (2, 18, 27, 34, 35, 71, 75). However, in contrast to the sustained checkpoint arrest of irradiated HR mutants, S. pombe Smc5-Smc6 hypomorphs, such as that with the smc6-74 mutation, enter highly aberrant mitoses following DNA damage. For DSBs induced by ionizing radiation, smc6 mutants progress into mitosis with wild-type kinetics, but, as shown by pulsed-field gel electrophoresis (PFGE), the chromosomes are highly fragmented (75). In each case, the mitotic defects are blocked by an earlier (upstream) HR defect (2, 27, 43). The chromosome segregation and recombination defects are apparent on each of the three S. pombe chromosomes and are not limited to the ribosomal DNA present on both ends of chromosome III.These aberrant mitoses of Smc5-Smc6 mutants following DNA damage either block segregation completely (the “cut” phenotype, where the division septum bisects the nucleus) or result in partially segregated chromosomes that are incompletely resolved along the division plane, with an elongated mitotic spindle. Since Smc5-Smc6 is required to maintain a damage induced checkpoint arrest, the aberrant mitoses of Smc5-Smc6 mutants could result from attempting to segregate incompletely repaired chromosomes. Alternatively, defects may reflect a role for Smc5-Smc6 in promoting chromosome segregation that is revealed in hypomorphic mutants following exogenous DNA damage but is evident in null mutants without DNA damage or with low-level endogenous lesions. Notably, while viable, the hypomorphic mutants show a high level of spontaneous aneuploidy, which is also consistent with defects in chromosome segregation (35, 75).Another characteristic of smc6 mutants in S. pombe is a strong synthetic lethality with a temperature-sensitive (ts) allele of topoisomerase II (Top2), top2-191, at a permissive temperature for top2-191 of 30°C. This lethality is due to a failure of chromosome segregation that resembles mitoses in irradiated smc6-74 cells (75). top2-191 is a A802V mutation (63), and cells with this mutation show no defects in cell cycle progression at 30°C. At 36°C, top2-191 cells enter mitosis with normal kinetics but fail to segregate chromosomes. The defects of top2-191 cells at the restrictive temperature of 36°C manifest exclusively in mitosis without an interphase delay and include defective chromosome condensation. Therefore, the top2-191 allele may not affect the postreplicative decatenation activity of Top2 in S. pombe. Rather, the smc6-top2-191 interaction may be related to the structural role played by Top2 in mitotic chromosome architecture (12, 14, 79).In vertebrate cells, defective decatenation caused by Top2 inhibition with drugs such as etoposide or doxorubicin block the rejoining of molecules cleaved by Top2. This leaves DSBs that elicit a G₂ DNA damage checkpoint response in many cell types (13, 16, 17, 38). Conversely, human cells in which Top2 has been deleted enter mitosis but show disordered chromosomes that fail to segregate (12). Thus, in S. pombe, top2-191 has a terminal phenotype more closely related to that of human cells with Top2 deleted than to that of cells with chemically inhibited Top2 that are blocked midway in the decatenation reaction.Here we have investigated the mitotic role of Smc5-Smc6 in S. pombe. We find that Smc5-Smc6 is required for the removal of cohesin from damaged chromosome arms prior to anaphase and from undamaged chromosomes when the mitotic function of Top2 is compromised. We show that a defect in cohesin removal is a major determinant of lethality in smc6 mutants and highlight the importance of coordinating Smc5-Smc6 and cohesin function in the maintenance of genome integrity.     

Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.     

Smc5-Smc6 complex suppresses gross chromosomal rearrangements mediated by break-induced replications

Translocations in chromosomes alter genetic information. Although the frequent translocations observed in many tumors suggest the altered genetic information by translocation could promote tumorigenesis, the mechanisms for how translocations are suppressed and produced are poorly understood. The smc6-9 mutation increased the translocation class gross chromosomal rearrangement (GCR). Translocations produced in the smc6-9 strain are unique because they are non-reciprocal and dependent on break-induced replication (BIR) and independent of non-homologous end joining. The high incidence of translocations near repetitive sequences such as delta sequences, ARS, tRNA genes, and telomeres in the smc6-9 strain indicates that Smc5-Smc6 suppresses translocations by reducing DNA damage at repetitive sequences. Synergistic enhancements of translocations in strains defective in DNA damage checkpoints by the smc6-9 mutation without affecting de novo telomere addition class GCR suggest that Smc5-Smc6 defines a new pathway to suppress GCR formation.     

The Smc5-Smc6 complex is required to remove chromosome junctions in meiosis

Meiosis, a specialized cell division with a single cycle of DNA replication round and two consecutive rounds of nuclear segregation, allows for the exchange of genetic material between parental chromosomes and the formation of haploid gametes. The structural maintenance of chromosome (SMC) proteins aid manipulation of chromosome structures inside cells. Eukaryotic SMC complexes include cohesin, condensin and the Smc5-Smc6 complex. Meiotic roles have been discovered for cohesin and condensin. However, although Smc5-Smc6 is known to be required for successful meiotic divisions, the meiotic functions of the complex are not well understood. Here we show that the Smc5-Smc6 complex localizes to specific chromosome regions during meiotic prophase I. We report that meiotic cells lacking Smc5-Smc6 undergo catastrophic meiotic divisions as a consequence of unresolved linkages between chromosomes. Surprisingly, meiotic segregation defects are not rescued by abrogation of Spo11-induced meiotic recombination, indicating that at least some chromosome linkages in smc5-smc6 mutants originate from other cellular processes. These results demonstrate that, as in mitosis, Smc5-Smc6 is required to ensure proper chromosome segregation during meiosis by preventing aberrant recombination intermediates between homologous chromosomes.     

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 Complex Regulates Recombination at Centromeric Regions and Affects Kinetochore Protein Sumoylation during Normal Growth

The Smc5-Smc6 complex in Saccharomyces cerevisiae is both essential for growth and important for coping with genotoxic stress. While it facilitates damage tolerance throughout the genome under genotoxin treatment, its function during unperturbed growth is mainly documented for repetitive DNA sequence maintenance. Here we provide physical and genetic evidence showing that the Smc5–Smc6 complex regulates recombination at non-repetitive loci such as centromeres in the absence of DNA damaging agents. Mutating Smc6 results in the accumulation of recombination intermediates at centromeres and other unique sequences as assayed by 2D gel analysis. In addition, smc6 mutant cells exhibit increased levels of Rad52 foci that co-localize with centromere markers. A rad52 mutation that decreases centromeric, but not overall, levels of Rad52 foci in smc6 mutants suppresses the nocodazole sensitivity of these cells, suggesting that the Smc6-mediated regulation of recombination at centromeric regions impacts centromere-related functions. In addition to influencing recombination, the SUMO ligase subunit of the Smc5–Smc6 complex promotes the sumoylation of two kinetochore proteins and affects mitotic spindles. These results suggest that the Smc5–Smc6 complex regulates both recombination and kinetochore sumoylation to facilitate chromosomal maintenance during growth.     

Dynamic and selective DNA-binding activity of Smc5, a core component of the Smc5-Smc6 complex

Members of the structural maintenance of chromosome (SMC) family of proteins are essential regulators of genomic stability. In particular, the conserved Smc5-6 complex is required for efficient DNA repair, checkpoint signaling, and DNA replication in all eukaryotes. Despite these important functions, the actual nature of the DNA substrates recognized by the Smc5-6 complex in chromosomes is currently unknown. Furthermore, how the core SMC components of the Smc5-6 complex use their ATPase-driven mechanochemical activities to act on chromosomes is not understood. Here, we address these issues by purifying and defining the DNA-binding activity of Smc5. We show that Smc5 binds strongly and specifically to single-stranded DNA (ssDNA). Remarkably, this DNA-binding activity is independent of Smc6 and is observed with the monomeric form of Smc5. We further show that Smc5 ATPase activity is essential for its functions in vivo and that ATP regulates the association of Smc5 with its substrates in vitro. Finally, we demonstrate that Smc5 is able to bind efficiently to oligonucleotides consistent in size with ssDNA intermediates produced during DNA replication and repair. Collectively, our data on the DNA-binding activities of Smc5 provide a compelling molecular basis for the role of the Smc5-6 complex in the DNA damage response.     

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.     

Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, postreplication repair (PRR) of UV-damaged DNA occurs by a Rad6-Rad18- and an Mms2-Ubc13-Rad5-dependent pathway or by a Rad52-dependent pathway. The Rad5 DNA helicase activity is specialized for promoting replication fork regression and template switching; previously, we suggested a role for the Rad5-dependent PRR pathway when the lesion is located on the leading strand and a role for the Rad52 pathway when the lesion is located on the lagging strand. In this study, we present evidence for the requirement of Nse1, a subunit of the Smc5-Smc6 complex, in Rad52-dependent PRR, and our genetic analyses suggest a role for the Nse1 and Mms21 E3 ligase activities associated with this complex in this repair mode. We discuss the possible ways by which the Smc5-Smc6 complex, including its associated ubiquitin ligase and SUMO ligase activities, might contribute to the Rad52-dependent nonrecombinational and recombinational modes of PRR.     

The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus

Homologous recombination (HR) is crucial for maintaining genome integrity by repairing DNA double-strand breaks (DSBs) and rescuing collapsed replication forks. In contrast, uncontrolled HR can lead to chromosome translocations, loss of heterozygosity, and deletion of repetitive sequences. Controlled HR is particularly important for the preservation of repetitive sequences of the ribosomal gene (rDNA) cluster. Here we show that recombinational repair of a DSB in rDNA in Saccharomyces cerevisiae involves the transient relocalization of the lesion to associate with the recombination machinery at an extranucleolar site. The nucleolar exclusion of Rad52 recombination foci entails Mre11 and Smc5-Smc6 complexes and depends on Rad52 SUMO (small ubiquitin-related modifier) modification. Remarkably, mutations that abrogate these activities result in the formation of Rad52 foci within the nucleolus and cause rDNA hyperrecombination and the excision of extrachromosomal rDNA circles. Our study also suggests a key role of sumoylation for nucleolar dynamics, perhaps in the compartmentalization of nuclear activities.     

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.     

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.     

Coordination of DNA damage responses via the Smc5/Smc6 complex

The detection of DNA damage activates DNA repair pathways and checkpoints to allow time for repair. Ultimately, these responses must be coordinated to ensure that cell cycle progression is halted until repair is completed. Several multiprotein complexes containing members of the structural maintenance of chromosomes family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization. Here we show that the Smc5/Smc6 (Smc5/6) complex is required for a coordinated response to DNA damage and normal chromosome integrity. Fission yeast cells lacking functional Smc6 initiate a normal checkpoint response to DNA damage, culminating in the phosphorylation and activation of the Chk1 protein kinase. Despite this, cells enter a lethal mitosis, presumably without completion of DNA repair. Another subunit of the complex, Nse1, is a conserved member of this complex and is also required for this response. We propose that the failure to maintain a checkpoint response stems from the lack of ongoing DNA repair or from defective chromosomal organization, which is the signal to maintain a checkpoint arrest. The Smc5/6 complex is fundamental to genome integrity and may function with the condensin and cohesin complexes in a coordinated manner.     

Smc5p promotes faithful chromosome transmission and DNA repair in Saccharomyces cerevisiae

Heterodimers of structural maintenance of chromosomes (SMC) proteins form the core of several protein complexes involved in the organization of DNA, including condensation and cohesion of the chromosomes at metaphase. The functions of the complexes with a heterodimer of Smc5p and Smc6p are less clear. To better understand them, we created two S. cerevisiae strains bearing temperature-sensitive alleles of SMC5. When shifted to the restrictive temperature, both mutants lose viability gradually, concomitant with the appearance of nuclear abnormalities and phosphorylation of the Rad53p DNA damage checkpoint protein. Removal of Rad52p or overexpression of the SUMO ligase Mms21p partially suppresses the temperature sensitivity of smc5 strains and increases their survival at the restrictive temperature. At the permissive temperature, smc5-31 but not smc5-33 cells exhibit hypersensitivity to several DNA-damaging agents despite induction of the DNA damage checkpoint. Similarly, smc5-31 but not smc5-33 cells are killed by overexpression of the SUMO ligase-defective Mms21-SAp but not by overexpression of wild-type Mms21p. Both smc5 alleles are synthetically lethal with mms21-SA and exhibit Rad52p-independent chromosome fragmentation and loss at semipermissive temperatures. Our data indicate a critical role for the S. cerevisiae Smc5/6-containing complexes in both DNA repair and chromosome segregation.     

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.     

Partners apart: Smc6-independent DNA binding activity of Smc5 on single-strand DNA

Comment on: Roy MA, et al. Cell Cycle 2011; 10:690-700.