Smc5-Smc6 Complex Research Articles

Structural mechanisms of SLF1 interactions with Histone H4 and RAD18 at the stalled replication fork.

DNA damage that obstructs the replication machinery poses a significant threat to genome stability. Replication-coupled repair mechanisms safeguard stalled replication forks by coordinating proteins involved in the DNA damage response (DDR) and replication. SLF1 (SMC5-SMC6 complex localization factor 1) is crucial for facilitating the recruitment of the SMC5/6 complex to damage sites through interactions with SLF2, RAD18,and nucleosomes. However, the structural mechanisms of SLF1's interactions are unclear. In this study, we determined the crystal structure of SLF1's ankyrin repeat domain bound to an unmethylated histone H4 tail, illustrating how SLF1 reads nascent nucleosomes. Using structure-based mutagenesis, we confirmed a phosphorylation-dependent interaction necessary for a stable complex between SLF1's tandem BRCA1 C-Terminal domain (tBRCT) and the phosphorylated C-terminal region (S442 and S444) of RAD18. We validated a functional role of conserved phosphate-binding residues inSLF1, and hydrophobic residues in RAD18 that areadjacent to phosphorylation sites, both of whichcontribute to the strong interaction. Interestingly, we discovered a DNA-binding property of this RAD18-binding interface, providing an additional domain of SLF1 to enhance binding to nucleosomes. Our results provide critical structural insights into SLF1's interactions with post-replicative chromatin and phosphorylation-dependent DDR signalling, enhancing our understanding of SMC5/6 recruitment and/or activity during replication-coupled DNA repair.

Genome control by SMC complexes.

Many cellular processes require large-scale rearrangements of chromatin structure. Structural maintenance of chromosomes (SMC) protein complexes are molecular machines that can provide structure to chromatin. These complexes can connect DNA elements in cis, walk along DNA, build and processively enlarge DNA loops and connect DNA molecules in trans to hold together the sister chromatids. These DNA-shaping abilities place SMC complexes at the heart of many DNA-based processes, including chromosome segregation in mitosis, transcription control and DNA replication, repair and recombination. In this Review, we discuss the latest insights into how SMC complexes such as cohesin, condensin and the SMC5-SMC6 complex shape DNA to direct these fundamental chromosomal processes. We also consider how SMC complexes, by building chromatin loops, can counteract the natural tendency of alike chromatin regions to cluster. SMC complexes thus control nuclear organization by participating in a molecular tug of war that determines the architecture of our genome.

The SMC5/6 complex compacts and silences unintegrated HIV-1 DNA and is antagonized by Vpr

SummarySilencing of nuclear DNA is an essential feature of innate immune responses to invading pathogens. Early in infection, unintegrated lentiviral cDNA accumulates in the nucleus yet remains poorly expressed. In HIV-1-like lentiviruses, the Vpr accessory protein enhances unintegrated viral DNA expression, suggesting Vpr antagonizes cellular restriction. We previously showed how Vpr remodels the host proteome, identifying multiple cellular targets. We now screen these using a targeted CRISPR-Cas9 library and identify SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1. SLF2 recruits the SMC5/6 complex to unintegrated lentiviruses, and depletion of SLF2, or the SMC5/6 complex, increases viral expression. ATAC-seq demonstrates that Vpr-mediated SLF2 depletion increases chromatin accessibility of unintegrated virus, suggesting that the SMC5/6 complex compacts viral chromatin to silence gene expression. This work implicates the SMC5/6 complex in nuclear immunosurveillance of extrachromosomal DNA and defines its targeting by Vpr as an evolutionarily conserved antagonism.

The SMC5/6 Complex Subunit NSE4A Is Involved in DNA Damage Repair and Seed Development.

The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.

Recruitment, loading, and activation of the Smc5-Smc6 SUMO ligase.

Duplication of the genome poses one of the most significant threats to genetic integrity, cellular fitness, and organismal health. Therefore, numerous mechanisms have evolved that maintain replication fork stability in the face of DNA damage and allow faithful genome duplication. The fission yeast BRCT-domain-containing protein Brc1, and its budding yeast orthologue Rtt107, has emerged as a "hub" factor that integrates multiple replication fork protection mechanisms. Notably, the cofactors and pathways through which Brc1, Rtt107, and their human orthologue (PTIP) acthave appeared largely distinct. This either represents true evolutionary functional divergence, or perhaps an incomplete genetic and biochemical analysis of each protein. In this regard, we recently showed that like Rtt107, Brc1 supports key functions of the Smc5-Smc6 complex, including its recruitment into DNA repair foci, chromatin association, and SUMO ligase activity. Furthermore, fission yeast cellslacking the Nse5-Nse6 genome stability factor were found to exhibit defects in Smc5-Smc6 function, similar to but more severe than those in cells lacking Brc1. Here, we place these findings in context with the known functions of Brc1, Rtt107, and Smc5-Smc6, present data suggesting a role for acetylation in Smc5-Smc6 chromatin loading, and discuss wider implications for genome stability.

155 Whole genome association analysis suggests an influence of inbreeding on bull sperm morphometry

Inbreeding depression, the genetic condition caused by mating related individuals, is particularly important in several cattle breeds with limited effective populations. This condition is often associated with decreases in performance, including fertility. Furthermore, sperm head morphometry was associated with fertility in several species. To our knowledge, the influence of inbreeding on sperm morphometry has not been widely reported in cattle. In this study, a Sperm Class Analyzer (SCA™, Microptic S.L., Barcelona, Spain) was used to characterise 7 sperm morphometry parameters in 59 Retinta bulls, including sperm head length, width, perimeter, ellipticity, elongation, regularity and rugosity. Two replicates of at least 100 sperm heads, from 2 frozen semen samples, were assessed per individual (n=200 sperm per bull). Additionally, all individuals were genotyped with the Axiom Bos1 high density SNP Array (Affymetrix, Santa Clara, CA, USA). The molecular-based inbreeding factor (FROH; mean 12.5%, range 1.75 to 33.0) had very low correlations with all sperm morphometry parameters. On average, sperm heads from bulls with higher FROH had a smaller area, but variability was high. Correlations between inbreeding and sperm shape were low and negative, length (r=−0.1449; P<0.01), width (r=−0.2494; P<0.01), and rugosity (r=−0.1086; P>0.01) being the most informative. Whole-genome association study (GWAS) analysis, performed using molecular inbreeding as co-factor, revealed 8 SNP, located on chromosomes BTA2, BTA5, BTA7, and BTA11, significantly associated with sperm head regularity and rugosity. Genomic analysis revealed that genes SLF1 and TMEFF2 are located close to enriched SNP. Gene SLF1 (SMC5-SMC6 complex localization factor 1) is associated with the regulation of protein complex and cytoskeleton assembly, whereas TMEFF2 (transmembrane protein with EGF like and 2 follistatin like domains 2) is associated with integral components of cell membrane and cell spreading and development. Therefore, we inferred that SLF1 and TMEFF2 may be involved in variations of sperm head shape. In this preliminary study, there was evidence of a potential influence of inbreeding on sperm morphometry in a beef cattle breed. However, additional studies, ideally including more individuals and additional breeds, are necessary to determine the validity of this potential association.

Retraction: Rtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids.

Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5–Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5–Smc6 complex is important for repair through the SCR pathway.

SMC complexes: from DNA to chromosomes.

SMC (structural maintenance of chromosomes) complexes - which include condensin, cohesin and the SMC5-SMC6 complex - are major components of chromosomes in all living organisms, from bacteria to humans. These ring-shaped protein machines, which are powered by ATP hydrolysis, topologically encircle DNA. With their ability to hold more than one strand of DNA together, SMC complexes control a plethora of chromosomal activities. Notable among these are chromosome condensation and sister chromatid cohesion. Moreover, SMC complexes have an important role in DNA repair. Recent mechanistic insight into the function and regulation of these universal chromosomal machines enables us to propose molecular models of chromosome structure, dynamics and function, illuminating one of the fundamental entities in biology.

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability.

Abstract 3301: Uncovering novel radiation sensitivity syndromes through exome sequencing

Abstract INTRODUCTION: The study of genetic disorders resulting in heightened sensitivity to ionizing radiation provides important molecular insights to DNA damage checkpoint and repair mechanisms. Individuals with these disorders have an increased risk of cancer and developmental abnormalities. We identified patients with unexplained radiation sensitivity disorders through the Texas Children's Cancer Center and Genetics Consult Service at Texas Children's Hospital. Children were screened for the known disorders prior to enrolling in a research study with whole exome sequencing. SUBJECTS: We identified three families with documented ionizing radiation sensitivity: (1) a sibling pair, one of whom had sternal osteomyelitis and a lung transplant (age 2) and a brother with eosinophilia and pneumonia (age 1); (2) a sibling pair, one child with astrocytoma (age 2) with severe leukoencephalopathy after radiation and a sister with medulloblastoma (age 2) and atypical encapsulated neuroma (age 13); and (3) a proband with leukemia and astrocytoma (age 2). All parents were unaffected. METHODS: We performed Illumina whole exome sequencing of probands and parents (when available), calling single nucleotide variants and insertions/deletions with ATLAS and PINDEL. We removed events >1% in 1079 unaffected normal samples, >1% dbSNP, or > = 20 individuals in Exome Aggregation Consortium. We kept rare missense and truncating variants, and prioritized rare compound heterozygous, homozygous, or mutations shared by affected sibs. RESULTS: In the proband with sternal osteomyelitis and lung transplant, we identified and validated compound heterozygous missense mutations in NDNL2 (MAGEG1), a small, intronless gene. NDNL2 is involved in the SMC5-SMC6 complex, and suggested to play a role in DNA repair. The known functions of NDNL2 may explain the finding of the patient's cells displaying altered phosphorylation upon DNA damage for NBN (absent) and SMC1 (reduced). We are currently investigating the link between mutant NDNL2 and phosphorylation of NBN and SMC1. In kindred 2 (previously shown to transmit a SMARCB1 splice site mutation), the siblings share rare compound heterozygous missense mutations in ROBO1, and a nonsense mutation in MEGF6. In kindred 3, the proband with leukemia and astrocytoma was known to carry a large Xp deletion, but we identified no obvious candidate underlying radiation sensitivity. CONCLUSION: Careful study of rare kindreds with unexplained radiation sensitivity may provide new insights to the DNA damage response. Interestingly, two families also carried a known genetic condition (SMARCB1 mutation and Xp microdeletion) that may explain cancer phenotype but does not appear to underlie radiation sensitivity. The very rare biallelic missense mutations in NDNL2 implicate it as a candidate radiation sensitivity gene to be validated by further functional testing. Both siblings in this family had severe infections resulting in death in early childhood. Citation Format: Deborah I. Ritter, Andrea K. Petersen, Katherine M. Haines, Ryan C. Zabriskie, David A. Wheeler, Sharon E. Plon. Uncovering novel radiation sensitivity syndromes through exome sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3301. doi:10.1158/1538-7445.AM2015-3301

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.

The Smc5-Smc6 heterodimer associates with DNA through several independent binding domains.

The Smc5-6 complex is required for the maintenance of genome integrity through its functions in DNA repair and chromosome biogenesis. However, the specific mode of action of Smc5 and Smc6 in these processes remains largely unknown. We previously showed that individual components of the Smc5-Smc6 complex bind strongly to DNA as monomers, despite the absence of a canonical DNA-binding domain (DBD) in these proteins. How heterodimerization of Smc5-6 affects its binding to DNA, and which parts of the SMC molecules confer DNA-binding activity is not known at present. To address this knowledge gap, we characterized the functional domains of the Smc5-6 heterodimer and identify two DBDs in each SMC molecule. The first DBD is located within the SMC hinge region and its adjacent coiled-coil arms, while the second is found in the conserved ATPase head domain. These DBDs can independently recapitulate the substrate preference of the full-length Smc5 and Smc6 proteins. We also show that heterodimerization of full-length proteins specifically increases the affinity of the resulting complex for double-stranded DNA substrates. Collectively, our findings provide critical insights into the structural requirements for effective binding of the Smc5-6 complex to DNA repair substrates in vitro and in live cells.

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.

Meiotic DNA joint molecule resolution depends on Nse5-Nse6 of the Smc5-Smc6 holocomplex

Faithful chromosome segregation in meiosis is crucial to form viable, healthy offspring and in most species, it requires programmed recombination between homologous chromosomes. In fission yeast, meiotic recombination is initiated by Rec12 (Spo11 homolog) and generates single Holliday junction (HJ) intermediates, which are resolved by the Mus81–Eme1 endonuclease to generate crossovers and thereby allow proper chromosome segregation. Although Mus81 contains the active site for HJ resolution, the regulation of Mus81–Eme1 is unclear. In cells lacking Nse5–Nse6 of the Smc5–Smc6 genome stability complex, we observe persistent meiotic recombination intermediates (DNA joint molecules) resembling HJs that accumulate in mus81Δ cells. Elimination of Rec12 nearly completely rescues the meiotic defects of nse6Δ and mus81Δ single mutants and partially rescues nse6Δ mus81Δ double mutants, indicating that these factors act after DNA double-strand break formation. Likewise, expression of the bacterial HJ resolvase RusA partially rescues the defects of nse6Δ, mus81Δ and nse6Δ mus81Δ mitotic cells, as well as the meiotic defects of nse6Δ and mus81Δ cells. Partial rescue likely reflects the accumulation of structures other than HJs, such as hemicatenanes, and an additional role for Nse5–Nse6 most prominent during mitotic growth. Our results indicate a regulatory role for the Smc5–Smc6 complex in HJ resolution via Mus81–Eme1.

SUMOylation of the α-Kleisin Subunit of Cohesin Is Required for DNA Damage-Induced Cohesion

Cohesion between sister chromatids is fundamental to ensure faithful chromosome segregation during mitosis and accurate repair of DNA damage postreplication. At the molecular level, cohesion establishment involves two defined events, a chromatin binding step and a chromatid entrapment event driven by posttranslational modifications on cohesin subunits. Here, we show that modification by the small ubiquitin-like protein (SUMO) is required for sister chromatid tethering after DNA damage. We find that all subunits of cohesin become SUMOylated upon exposure to DNA damaging agents or presence of a DNA double-strand break. We have mapped all lysine residues on cohesin's α-kleisin subunit Mcd1 (Scc1) where SUMO can conjugate. We demonstrate that Mcd1 SUMOylation-deficient alleles are still recruited to DSB-proximal regions but are defective in tethering sister chromatids and consequently fail to establish damage-induced cohesion both at DSBs and undamaged chromosomes. Moreover, we demonstrate that the bulk of Mcd1 SUMOylation in response to damage is carried out by the SUMO E3 ligase Nse2, a subunit of the related Smc5-Smc6 complex. SUMOylation occurs in cells with compromised Chk1 kinase activity, necessary for known posttranslational modifications on Mcd1, required for damage-induced cohesion. These findings demonstrate that SUMOylation of Mcd1 is a novel prerequisite for the establishment of DNA damage-induced cohesion at DSB-proximal regions and cohesion-associating regions (CARs) genome-wide.

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.

Rtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids

Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5–Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5–Smc6 complex is important for repair through the SCR pathway.

Double-Strand Breaks in Heterochromatin Move Outside of a Dynamic HP1a Domain to Complete Recombinational Repair

Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats.

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.

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.