Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans

Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1. Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1. We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability.

[1]  S. Gabriel,et al.  A Dominant Mutation in Human RAD51 Reveals Its Function in DNA Interstrand Crosslink Repair Independent of Homologous Recombination. , 2015, Molecular cell.

[2]  W. Heyer Regulation of recombination and genomic maintenance. , 2015, Cold Spring Harbor Perspectives in Biology.

[3]  P. Sung,et al.  Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52 , 2015, Nature Communications.

[4]  E. Egelman,et al.  Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination , 2015, Cell.

[5]  M. Jasin,et al.  Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins. , 2015, Cold Spring Harbor perspectives in biology.

[6]  Stephen K. Godin,et al.  Evolutionary and Functional Analysis of the Invariant SWIM Domain in the Conserved Shu2/SWS1 Protein Family from Saccharomyces cerevisiae to Homo sapiens , 2015, Genetics.

[7]  M. Krause,et al.  Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans , 2014, Genetics.

[8]  Joshua A. Arribere,et al.  Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans , 2014, Genetics.

[9]  J. Yanowitz,et al.  Methodological considerations for mutagen exposure in C. elegans. , 2014, Methods.

[10]  Benjamin Lant,et al.  Fluorescent visualization of germline apoptosis in living Caenorhabditis elegans. , 2014, Cold Spring Harbor protocols.

[11]  Soogil Hong,et al.  Shu1 promotes homolog bias of meiotic recombination in Saccharomyces cerevisiae , 2013, Molecules and cells.

[12]  Rodney Rothstein,et al.  Repair of strand breaks by homologous recombination. , 2013, Cold Spring Harbor perspectives in biology.

[13]  Bob Goldstein,et al.  Engineering the Caenorhabditis elegans Genome Using Cas9-Triggered Homologous Recombination , 2013, Nature Methods.

[14]  N. Kleckner,et al.  The logic and mechanism of homologous recombination partner choice. , 2013, Molecular cell.

[15]  H. Hosaka,et al.  A new protein complex promoting the assembly of Rad51 filaments , 2013, Nature Communications.

[16]  Adam D. Wier,et al.  The Shu complex interacts with Rad51 through the Rad51 paralogues Rad55–Rad57 to mediate error-free recombination , 2013, Nucleic acids research.

[17]  Kara A. Bernstein,et al.  From yeast to mammals: recent advances in genetic control of homologous recombination. , 2012, DNA repair.

[18]  E. Shtykova,et al.  Structural and SAXS analysis of the budding yeast SHU‐complex proteins , 2012, FEBS letters.

[19]  T. Hashimshony,et al.  A genomic bias for genotype–environment interactions in C. elegans , 2012, Molecular systems biology.

[20]  Xu Li,et al.  Structural Analysis of Shu Proteins Reveals a DNA Binding Role Essential for Resisting Damage* , 2012, The Journal of Biological Chemistry.

[21]  Xiaolan Zhao,et al.  Homologous recombination and its regulation , 2012, Nucleic acids research.

[22]  M. Tarsounas,et al.  RAD51 paralogs: roles in DNA damage signalling, recombinational repair and tumorigenesis. , 2011, Seminars in cell & developmental biology.

[23]  Junjie Chen,et al.  hSWS1·SWSAP1 Is an Evolutionarily Conserved Complex Required for Efficient Homologous Recombination Repair* , 2011, The Journal of Biological Chemistry.

[24]  S. Keeney,et al.  Evolutionary conservation of meiotic DSB proteins: more than just Spo11. , 2010, Genes & development.

[25]  Franca Fraternali,et al.  Mutation of the RAD51C gene in a Fanconi anemia–like disorder , 2010, Nature Genetics.

[26]  P. Plevani,et al.  Overlapping mechanisms promote postsynaptic RAD-51 filament disassembly during meiotic double-strand break repair. , 2010, Molecular cell.

[27]  W. Xiao,et al.  The yeast Shu complex couples error‐free post‐replication repair to homologous recombination , 2009, Molecular microbiology.

[28]  D. Haines,et al.  Loss of Rad51c leads to embryonic lethality and modulation of Trp53-dependent tumorigenesis in mice. , 2009, Cancer research.

[29]  P. Plevani,et al.  Caenorhabditis elegans POLQ-1 and HEL-308 function in two distinct DNA interstrand cross-link repair pathways. , 2008, DNA repair.

[30]  Thomas D. Schmittgen,et al.  Analyzing real-time PCR data by the comparative CT method , 2008, Nature Protocols.

[31]  J. Yanowitz Genome Integrity Is Regulated by the Caenorhabditis elegans Rad51D Homolog rfs-1 , 2008, Genetics.

[32]  J. Vandesompele,et al.  Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans , 2008, BMC Molecular Biology.

[33]  I. Hickson,et al.  Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3. , 2007, Molecular biology of the cell.

[34]  A. Villeneuve,et al.  SYP-3 Restricts Synaptonemal Complex Assembly to Bridge Paired Chromosome Axes During Meiosis in Caenorhabditis elegans , 2007, Genetics.

[35]  A. Villeneuve,et al.  Synapsis-Defective Mutants Reveal a Correlation Between Chromosome Conformation and the Mode of Double-Strand Break Repair During Caenorhabditis elegans Meiosis , 2007, Genetics.

[36]  A. Villeneuve,et al.  Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line. , 2007, Developmental biology.

[37]  S. Boulton,et al.  Replication blocking lesions present a unique substrate for homologous recombination , 2007, The EMBO journal.

[38]  James H. Thomas,et al.  Mutator Phenotype of Caenorhabditis elegans DNA Damage Checkpoint Mutants , 2006, Genetics.

[39]  J. Yates,et al.  Sws1 is a conserved regulator of homologous recombination in eukaryotic cells , 2006, The EMBO journal.

[40]  N. O'Neil,et al.  Homologous Recombination Is Required for Genome Stability in the Absence of DOG-1 in Caenorhabditis elegans , 2006, Genetics.

[41]  J. Thacker The RAD51 gene family, genetic instability and cancer. , 2005, Cancer letters.

[42]  R. Rothstein,et al.  A Genetic Screen for top3 Suppressors in Saccharomyces cerevisiae Identifies SHU1, SHU2, PSY3 and CSM2 , 2005, Genetics.

[43]  A. Villeneuve,et al.  Chromosome-Wide Control of Meiotic Crossing over in C. elegans , 2003, Current Biology.

[44]  J. Bessereau,et al.  [C. elegans: of neurons and genes]. , 2003, Medecine sciences : M/S.

[45]  A. Gartner,et al.  Genetic and cytological characterization of the recombination protein RAD-51 in Caenorhabditis elegans , 2003, Chromosoma.

[46]  P. Meneely,et al.  Crossover distribution and high interference for both the X chromosome and an autosome during oogenesis and spermatogenesis in Caenorhabditis elegans. , 2002, Genetics.

[47]  Iris Cheung,et al.  Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA , 2002, Nature Genetics.

[48]  E. Koonin,et al.  SWIM, a novel Zn-chelating domain present in bacteria, archaea and eukaryotes. , 2002, Trends in biochemical sciences.

[49]  Joanna S Albala,et al.  RAD51C Interacts with RAD51B and Is Central to a Larger Protein Complex in Vivo Exclusive of RAD51* , 2002, The Journal of Biological Chemistry.

[50]  J. Thacker,et al.  Xrcc2 is required for genetic stability, embryonic neurogenesis and viability in mice , 2000, The EMBO journal.

[51]  S. Keeney,et al.  Meiosis-Specific DNA Double-Strand Breaks Are Catalyzed by Spo11, a Member of a Widely Conserved Protein Family , 1997, Cell.

[52]  E. Craig,et al.  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. , 1996, Genetics.

[53]  A. Coulson,et al.  Meiotic recombination, noncoding DNA and genomic organization in Caenorhabditis elegans. , 1995, Genetics.

[54]  N. Munakata [Genetics of Caenorhabditis elegans]. , 1989, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[55]  S. Brenner,et al.  Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS. , 1979, Genetics.

[56]  A. Gartner,et al.  Methods for studying the DNA damage response in the Caenorhabdatis elegans germ line. , 2012, Methods in cell biology.

[57]  M. Krause,et al.  Myogenic conversion and transcriptional profiling of embryonic blastomeres in Caenorhabditis elegans. , 2012, Methods.