Chromosome Instability and Defective Recombinational Repair in Knockout Mutants of the Five Rad51 Paralogs

ABSTRACT The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.

[1]  D. Pinkel,et al.  Measurement of sister chromatid exchanges at very low bromodeoxyuridine substitution levels using a monoclonal antibody in Chinese hamster ovary cells. , 1985, Cancer research.

[2]  A. Carrano,et al.  Cytogenetic characterization of the ionizing radiation-sensitive Chinese hamster mutant irs1. , 1991, Mutation research.

[3]  Jean-Marie Buerstedde,et al.  Increased ratio of targeted to random integration after transfection of chicken B cell lines , 1991, Cell.

[4]  L. Hartwell,et al.  Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae. , 1992, Genetics.

[5]  K. Ikeo,et al.  Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA , 1993, Nature Genetics.

[6]  S. West,et al.  Resolution of holliday junctions by RuvC resolvase: Cleavage specificity and DNA distortion , 1993, Cell.

[7]  X. Wang,et al.  Loss of S-phase-dependent radioresistance in irs-1 cells exposed to X-rays. , 1994, Mutation research.

[8]  D. K. Bishop.  RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis , 1994, Cell.

[9]  A. Shinohara,et al.  Homologous recombination and the roles of double-strand breaks. , 1995, Trends in biochemical sciences.

[10]  L. Symington,et al.  Copyright � 1995, American Society for Microbiology Functional Differences and Interactions among the Putative , 1995 .

[11]  R. Tebbs,et al.  Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  S. Lippard,et al.  Cisplatin and DNA repair in cancer chemotherapy. , 1995, Trends in biochemical sciences.

[13]  P. Berg,et al.  Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  P. Hasty,et al.  A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53 , 1996, Molecular and cellular biology.

[15]  G. Wahl,et al.  Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Julian Peto,et al.  Identification of the breast cancer susceptibility gene BRCA2 , 1996, Nature.

[17]  Y. Nishimune,et al.  The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis. , 1996, Nucleic acids research.

[18]  N. Kamada,et al.  S phase specific formation of the human Rad51 protein nuclear foci in lymphocytes. , 1996, Oncogene.

[19]  K. Nakao,et al.  Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Yuko Yamaguchi-Iwai,et al.  Reduced X-Ray Resistance and Homologous Recombination Frequencies in a RAD54−/− Mutant of the Chicken DT40 Cell Line , 1997, Cell.

[21]  Ralph Scully,et al.  Dynamic Changes of BRCA1 Subnuclear Location and Phosphorylation State Are Initiated by DNA Damage , 1997, Cell.

[22]  J. Hoeijmakers,et al.  Disruption of Mouse RAD54 Reduces Ionizing Radiation Resistance and Homologous Recombination , 1997, Cell.

[23]  J. Albala,et al.  Identification of a novel human RAD51 homolog, RAD51B. , 1997, Genomics.

[24]  E. Gordon-Smith,et al.  The sensitivity of Fanconi anaemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA crosslinking , 1997, British journal of haematology.

[25]  L. Bennett,et al.  Characterization of the rat and mouse homologues of the BRCA2 breast cancer susceptibility gene. , 1997, Cancer research.

[26]  M. Meyn.  Chromosome instability syndromes: lessons for carcinogenesis. , 1997, Current topics in microbiology and immunology.

[27]  D. Weaver,et al.  Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells. , 1997, Nucleic acids research.

[28]  L. Hartwell,et al.  Integrating genetic approaches into the discovery of anticancer drugs. , 1997, Science.

[29]  P. Sung.  Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. , 1997, Genes & development.

[30]  F. Bullrich,et al.  Isolation of human and mouse genes based on homology to REC2, a recombinational repair gene from the fungus Ustilago maydis. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Hoeijmakers,et al.  Molecular mechanisms of DNA double strand break repair. , 1998, Trends in cell biology.

[32]  B. Morolli,et al.  Targeted Inactivation of Mouse RAD52Reduces Homologous Recombination but Not Resistance to Ionizing Radiation , 1998, Molecular and Cellular Biology.

[33]  J. Thacker,et al.  Isolation of novel human and mouse genes of the recA/RAD51 recombination-repair gene family. , 1998, Nucleic acids research.

[34]  Stephen L. Gasior,et al.  Rad52 associates with RPA and functions with rad55 and rad57 to assemble meiotic recombination complexes. , 1998, Genes & development.

[35]  Y. Yamaguchi-Iwai,et al.  Homologous recombination and non‐homologous end‐joining pathways of DNA double‐strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells , 1998, The EMBO journal.

[36]  Richard Cartwright,et al.  The XRCC2 DNA repair gene from human and mouse encodes a novel member of the recA/RAD51 family , 1998, Nucleic Acids Res..

[37]  Akira Shinohara,et al.  Rad51‐deficient vertebrate cells accumulate chromosomal breaks prior to cell death , 1998, The EMBO journal.

[38]  P. Baumann,et al.  Role of the human RAD51 protein in homologous recombination and double-stranded-break repair. , 1998, Trends in biochemical sciences.

[39]  S. Kowalczykowski,et al.  DNA strand exchange proteins: a biochemical and physical comparison. , 1998, Frontiers in bioscience : a journal and virtual library.

[40]  M. Kawabata,et al.  Sequence analysis and expression of a novel mouse homolog of Escherichia coli recA gene. , 1998, Biochimica et biophysica acta.

[41]  B. Ponder,et al.  Involvement of Brca2 in DNA repair. , 1998, Molecular cell.

[42]  Y. Chen,et al.  The BRC repeats in BRCA2 are critical for RAD51 binding and resistance to methyl methanesulfonate treatment. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[43]  J. Schimenti,et al.  Identification, characterization, and genetic mapping of Rad51d, a new mouse and human RAD51/RecA-related gene. , 1998, Genomics.

[44]  J. Albala,et al.  Isolation and characterization of RAD51C, a new human member of the RAD51 family of related genes. , 1998, Nucleic acids research.

[45]  F. Couch,et al.  Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. , 1998, Molecular cell.

[46]  R. Weichselbaum,et al.  Xrcc3 Is Required for Assembly of Rad51 Complexes in Vivo * , 1998, The Journal of Biological Chemistry.

[47]  J. Lamerdin,et al.  XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages. , 1998, Molecular cell.

[48]  M. Jasin,et al.  Homology-directed repair is a major double-strand break repair pathway in mammalian cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Akira Shinohara,et al.  Homologous Recombination, but Not DNA Repair, Is Reduced in Vertebrate Cells Deficient in RAD52 , 1998, Molecular and Cellular Biology.

[50]  G E Tomlinson,et al.  BRCA2 is required for ionizing radiation-induced assembly of Rad51 complex in vivo. , 1999, Cancer research.

[51]  W. V. D. Van de Ven,et al.  Allelic knockout of novel splice variants of human recombination repair gene RAD51B in t(12;14) uterine leiomyomas. , 1999, Cancer research.

[52]  M. Jasin,et al.  Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination , 1999, Nature.

[53]  T Yagi,et al.  Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells , 1999, The EMBO journal.

[54]  D. Ward,et al.  Sequestration of Mammalian Rad51-Recombination Protein into Micronuclei , 1999, The Journal of cell biology.

[55]  N. Maizels,et al.  Localization and dynamic relocalization of mammalian Rad52 during the cell cycle and in response to DNA damage , 1999, Current Biology.

[56]  J. Thacker.  A surfeit of RAD51-like genes? , 1999, Trends in Genetics.

[57]  D. Schild,et al.  The contribution of homologous recombination in preserving genome integrity in mammalian cells. , 1999, Biochimie.

[58]  S. Kathiresan,et al.  hREC2, a RAD51-like gene, is disrupted by t(12;14) (q15;q24.1) in a uterine leiomyoma. , 1999, Cancer genetics and cytogenetics.

[59]  J. Haber.  DNA recombination: the replication connection. , 1999, Trends in biochemical sciences.

[60]  T. Haaf,et al.  Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[61]  Yuko Yamaguchi-Iwai,et al.  Sister Chromatid Exchanges Are Mediated by Homologous Recombination in Vertebrate Cells , 1999, Molecular and Cellular Biology.

[62]  K. Yoshioka,et al.  Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation , 1999, Oncogene.

[63]  L. Thompson,et al.  XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. , 1999, Genes & development.

[64]  M. Ehrlich.  DNA alterations in cancer : genetic and epigenetic changes , 2000 .

[65]  N. Maizels,et al.  Coordinated response of mammalian Rad51 and Rad52 to DNA damage , 2000, EMBO reports.

[66]  M. Jasin,et al.  Sister chromatid gene conversion is a prominent double‐strand break repair pathway in mammalian cells , 2000, The EMBO journal.

[67]  S. West,et al.  Gross chromosomal rearrangements and genetic exchange between nonhomologous chromosomes following BRCA2 inactivation. , 2000, Genes & development.

[68]  D. J. Chen,et al.  XRCC3 is required for efficient repair of chromosome breaks by homologous recombination. , 2000, Mutation research.

[69]  S. Kowalczykowski.  Initiation of genetic recombination and recombination-dependent replication. , 2000, Trends in biochemical sciences.

[70]  T. Katada,et al.  Possible association of BLM in decreasing DNA double strand breaks during DNA replication , 2000, The EMBO journal.

[71]  N. Kamada,et al.  Rad51 Accumulation at Sites of DNA Damage and in Postreplicative Chromatin , 2000, The Journal of cell biology.

[72]  P. Sung,et al.  Tailed duplex DNA is the preferred substrate for Rad 51 protein-mediated homologous pairing preference for 3-end invasion is generally considered , 2000 .

[73]  J. Braybrooke,et al.  The RAD51 Family Member, RAD51L3, Is a DNA-stimulated ATPase That Forms a Complex with XRCC2* , 2000, The Journal of Biological Chemistry.

[74]  David J. Chen,et al.  Evidence for Simultaneous Protein Interactions between Human Rad51 Paralogs* , 2000, The Journal of Biological Chemistry.

[75]  J. Albala,et al.  The Rad51 Paralog Rad51B Promotes Homologous Recombinational Repair , 2000, Molecular and Cellular Biology.

[76]  D. Schild,et al.  Homologous recombinational repair of DNA ensures mammalian chromosome stability. , 2001, Mutation research.