SMC5 and SMC6 genes are required for the segregation of repetitive chromosome regions

Structure chromosome (SMC) proteins organize the core of cohesin, condensin and Smc5-Smc6 complexes. The Smc5-Smc6 complex is required for DNA repair, as well as having another essential but enigmatic function. Here, we generated conditional mutants of SMC5 and SMC6 in budding yeast, in which the essential function was affected. We show that mutant smc5-6 and smc6-9 cells undergo an aberrant mitosis in which chromosome segregation of repetitive regions is impaired; this leads to DNA damage and RAD9-dependent activation of the Rad53 protein kinase. Consistent with a requirement for the segregation of repetitive regions, Smc5 and Smc6 proteins are enriched at ribosomal DNA (rDNA) and at some telomeres. We show that, following Smc5-Smc6 inactivation, metaphase-arrested cells show increased levels of X-shaped DNA (Holliday junctions) at the rDNA locus. Furthermore, deletion of RAD52 partially suppresses the temperature sensitivity of smc5-6 and smc6-9 mutants. We also present evidence showing that the rDNA segregation defects of smc5/smc6 mutants are mechanistically different from those previously observed for condensin mutants. These results point towards a role for the Smc5-Smc6 complex in preventing the formation of sister chromatid junctions, thereby ensuring the correct partitioning of chromosomes during anaphase.     

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.     

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.     

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.     

SMC6 is required for MMS-induced interchromosomal and sister chromatid recombinations in Saccharomyces cerevisiae

SMC6 (RHC18) in Saccharomyces cerevisiae, which is a homologue of the Schizosaccharomyces pombe rad18+ gene and essential for cell viability, encodes a structural maintenance of chromosomes (SMC) family protein. In contrast to the rest of the SMC family of proteins, Smc1-Smc4, which are the components of cohesin or condensin, little is known about Smc6. In this study, we generated temperature sensitive (ts) smc6 mutants of budding yeast and characterized their properties. One ts-mutant, smc6-56, ceased growth soon after up-shift to a non-permissive temperature, arrested in the late S and G2/M phase, and gradually lost viability. smc6-56 cells at a permissive temperature showed a higher sensitivity than wild-type cells to various DNA damaging agents including methyl methanesulfonate (MMS). The rad52 smc6-56 double mutant showed a sensitivity to MMS similar to that of the rad52 single mutant, indicating that Smc6 is involved in a pathway that requires Rad52 to function. Moreover, no induction of interchromosomal recombination and sister chromatid recombination was observed in smc6-56 cells, which occurred in wild-type cells upon exposure to MMS.     

Homologous recombination-dependent rescue of deficiency in the structural maintenance of chromosomes (Smc) 5/6 complex

The essential and evolutionarily conserved Smc5-Smc6 complex (Smc5/6) is critical for the maintenance of genome stability. Partial loss of Smc5/6 function yields several defects in DNA repair, which are rescued by inactivation of the homologous recombination (HR) machinery. Thus HR is thought to be toxic to cells with defective Smc5/6. Recent work has highlighted a role for Smc5/6 and the Sgs1 DNA helicase in preventing the accumulation of unresolved HR intermediates. Here we investigate how deletion of MPH1, encoding the orthologue of the human FANCM DNA helicase, rescues the DNA damage sensitivity of smc5/6 but not sgs1Δ mutants. We find that MPH1 deletion diminishes accumulation of HR intermediates within both smc5/6 and sgs1Δ cells, suggesting that MPH1 deletion is sufficient to decrease the use of template switch recombination (TSR) to bypass DNA lesions. We further explain how avoidance of TSR is nonetheless insufficient to rescue defects in sgs1Δ mutants, by demonstrating a requirement for Sgs1, along with the post-replicative repair (PRR) and HR machinery, in a pathway that operates in mph1Δ mutants. In addition, we map the region of Mph1 that binds Smc5, and describe a novel allele of MPH1 encoding a protein unable to bind Smc5 (mph1-Δ60). Remarkably, mph1-Δ60 supports normal growth and responses to DNA damaging agents, indicating that Smc5/6 does not simply restrain the recombinogenic activity of Mph1 via direct binding. These data as a whole highlight a role for Smc5/6 and Sgs1 in the resolution of Mph1-dependent HR intermediates.     

Smc5-Smc6 Complex Preserves Nucleolar Integrity in S. cerevisiae

As a baton in a relay race, intact genomes need to be smoothly passed onto daughter cells every cell generation. Cohesin and condensin are multiprotein complexes involved in chromosome segregation during mitosis, they perform the crucial function of organizing and compacting chromosomes into pairs to facilitate their equal distribution in anaphase. Both complexes share a core of similar origin, containing a heterodimer formed by members of the conserved chromosomal ATPase family named Smc. A third complex containing Smc proteins at its core, the Smc5-Smc6 complex, previously known to be involved in DNA repair has recently been shown to contribute to chromosome segregation during anaphase. Smc5-Smc6 plays a role in the disjunction of repetitive regions. Here, we present results further supporting the importance of Smc5-Smc6 in maintaining the integrity of the repetitive ribosomal DNA (rDNA) locus, the largest repetitive region of the budding yeast genome.     

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.     

Brc1-mediated rescue of Smc5/6 deficiency: requirement for multiple nucleases and a novel Rad18 function

Smc5/6 is a structural maintenance of chromosomes complex, related to the cohesin and condensin complexes. Recent studies implicate Smc5/6 as being essential for homologous recombination. Each gene is essential, but hypomorphic alleles are defective in the repair of a diverse array of lesions. A particular allele of smc6 (smc6-74) is suppressed by overexpression of Brc1, a six-BRCT domain protein that is required for DNA repair during S-phase. This suppression requires the postreplication repair (PRR) protein Rhp18 and the structure-specific endonucleases Slx1/4 and Mus81/Eme1. However, we show here that the contribution of Rhp18 is via a novel pathway that is independent of PCNA ubiquitination and PRR. Moreover, we identify Exo1 as an additional nuclease required for Brc1-mediated suppression of smc6-74, independent of mismatch repair. Further, the Apn2 endonuclease is required for the viability of smc6 mutants without extrinsic DNA damage, although this is not due to a defect in base excision repair. Several nucleotide excision repair genes are similarly shown to ensure viability of smc6 mutants. The requirement for excision factors for the viability of smc6 mutants is consistent with an inability to respond to spontaneous lesions by Smc5/6-dependent recombination.     

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.     

Brc1-mediated DNA repair and damage tolerance

The structural maintenance of chromosome (SMC) proteins are key elements in controlling chromosome dynamics. In eukaryotic cells, three essential SMC complexes have been defined: cohesin, condensin, and the Smc5/6 complex. The latter is essential for DNA damage responses; in its absence both repair and checkpoint responses fail. In fission yeast, the UV-C and ionizing radiation (IR) sensitivity of a specific hypomorphic allele encoding the Smc6 subunit, rad18-74 (renamed smc6-74), is suppressed by mild overexpression of a six-BRCT-domain protein, Brc1. Deletion of brc1 does not result in a hypersensitivity to UV-C or IR, and thus the function of Brc1 relative to the Smc5/6 complex has remained unclear. Here we show that brc1Delta cells are hypersensitive to a range of radiomimetic drugs that share the feature of creating lesions that are an impediment to the completion of DNA replication. Through a genetic analysis of brc1Delta epistasis and by defining genes required for Brc1 to suppress smc6-74, we find that Brc1 functions to promote recombination through a novel postreplication repair pathway and the structure-specific nucleases Slx1 and Mus81. Activation of this pathway through overproduction of Brc1 bypasses a repair defect in smc6-74, reestablishing resolution of lesions by recombination.     

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.     

Incision of damaged DNA in the presence of an impaired Smc5/6 complex imperils genome stability

The Smc5/6 complex is implicated in homologous recombination-mediated DNA repair during DNA damage or replication stress. Here, we analysed genome-wide replication dynamics in a hypomorphic budding yeast mutant, smc6-P4. The overall replication dynamics in the smc6 mutant is similar to that in the wild-type cells. However, we captured a difference in the replication profile of an early S phase sample in the mutant, prompting the hypothesis that the mutant incorporates ribonucleotides and/or accumulates single-stranded DNA gaps during replication. We tested if inhibiting the ribonucleotide excision repair pathway would exacerbate the smc6 mutant in response to DNA replication stress. Contrary to our expectation, impairment of ribonucleotide excision repair, as well as virtually all other DNA repair pathways, alleviated smc6 mutant''s hypersensitivity to induced replication stress. We propose that nucleotide incision in the absence of a functional Smc5/6 complex has more disastrous outcomes than the damage per se. Our study provides novel perspectives for the role of the Smc5/6 complex during DNA replication.     

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.     

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.     

Roles of vertebrate Smc5 in sister chromatid cohesion and homologous recombinational repair

The structural maintenance of chromosomes (Smc) family members Smc5 and Smc6 are both essential in budding and fission yeasts. Yeast smc5/6 mutants are hypersensitive to DNA damage, and Smc5/6 is recruited to HO-induced double-strand breaks (DSBs), facilitating intersister chromatid recombinational repair. To determine the role of the vertebrate Smc5/6 complex during the normal cell cycle, we generated an Smc5-deficient chicken DT40 cell line using gene targeting. Surprisingly, Smc5(-) cells were viable, although they proliferated more slowly than controls and showed mitotic abnormalities. Smc5-deficient cells were sensitive to methyl methanesulfonate and ionizing radiation (IR) and showed increased chromosome aberration levels upon irradiation. Formation and resolution of Rad51 and gamma-H2AX foci after irradiation were altered in Smc5 mutants, suggesting defects in homologous recombinational (HR) repair of DNA damage. Ku70(-/-) Smc5(-) cells were more sensitive to IR than either single mutant, with Rad54(-/-) Smc5(-) cells being no more sensitive than Rad54(-/-) cells, consistent with an HR function for the vertebrate Smc5/6 complex. Although gene targeting occurred at wild-type levels, recombinational repair of induced double-strand breaks was reduced in Smc5(-) cells. Smc5 loss increased sister chromatid exchanges and sister chromatid separation distances in mitotic chromosomes. We conclude that Smc5/6 regulates recombinational repair by ensuring appropriate sister chromatid cohesion.     

Msh2 Blocks an Alternative Mechanism for Non-Homologous Tail Removal during Single-Strand Annealing in Saccharomyces cerevisiae

Chromosomal translocations are frequently observed in cells exposed to agents that cause DNA double-strand breaks (DSBs), such as ionizing radiation and chemotherapeutic drugs, and are often associated with tumors in mammals. Recently, translocation formation in the budding yeast, Saccharomyces cerevisiae, has been found to occur at high frequencies following the creation of multiple DSBs adjacent to repetitive sequences on non-homologous chromosomes. The genetic control of translocation formation and the chromosome complements of the clones that contain translocations suggest that translocation formation occurs by single-strand annealing (SSA). Among the factors important for translocation formation by SSA is the central mismatch repair (MMR) and homologous recombination (HR) factor, Msh2. Here we describe the effects of several msh2 missense mutations on translocation formation that suggest that Msh2 has separable functions in stabilizing annealed single strands, and removing non-homologous sequences from their ends. Additionally, interactions between the msh2 alleles and a null allele of RAD1, which encodes a subunit of a nuclease critical for the removal of non-homologous tails suggest that Msh2 blocks an alternative mechanism for removing these sequences. These results suggest that Msh2 plays multiple roles in the formation of chromosomal translocations following acute levels of DNA damage.     

Rad50 is involved in MMS-induced recombination between homologous chromosomes in mitotic cells

The structural maintenance of chromosomes (SMC) family proteins (Smc1-Smc6) typically consist of two coiled-coil domains, an amino-terminal head domain, and a carboxyl-terminal tail domain. Rad50, a component of the Mre11/Rad50/Xrs2 (MRX) complex, has a similar domain structure to the SMC proteins. In Saccharomyces cerevisiae, the MRX complex appears to be essential for recombination between homologous chromosomes in meiotic cells, but not in cells undergoing vegetative growth. Here we provide for the first time evidence that Rad50, like Smc6, is required for the induction of recombination between homologous chromosomes in cells in the vegetative growth state upon exposure to methyl methanesulfonate. However, UV-induced recombination between homologous chromosomes is intact in both rad50 and smc6-56 mutant cells.     

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.