Purified human BRCA2 stimulates RAD51-mediated recombination

Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report the purification of full-length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by targeting RAD51 to ssDNA over double-stranded DNA, enabling RAD51 to displace replication protein-A (RPA) from ssDNA and stabilizing RAD51–ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer.

[1]  P. Baumann,et al.  Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52. , 1999, Journal of molecular biology.

[2]  S. Kowalczykowski,et al.  Escherichia coli RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein: A common step in genetic recombination , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[3]  S. West,et al.  Interactions between human BRCA2 protein and the meiosis‐specific recombinase DMC1 , 2007, The EMBO journal.

[4]  S. Kowalczykowski,et al.  BRCA2: Shining light on the regulation of DNA-binding selectivity by RAD51 , 2009, Cell cycle.

[5]  H. Joenje,et al.  Impaired DNA damage-induced nuclear Rad51 foci formation uniquely characterizes Fanconi anemia group D1 , 2002, Oncogene.

[6]  K. Kinzler,et al.  Genetic instabilities in human cancers , 1998, Nature.

[7]  N. Pavletich,et al.  The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA–ssDNA junction , 2005, Nature.

[8]  Phang-lang Chen,et al.  Expression of BRC Repeats in Breast Cancer Cells Disrupts the BRCA2-Rad51 Complex and Leads to Radiation Hypersensitivity and Loss of G2/M Checkpoint Control* , 1999, The Journal of Biological Chemistry.

[9]  Jeremy M. Stark,et al.  Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[10]  S. Kowalczykowski,et al.  The DNA Binding Properties of Saccharomyces cerevisiae Rad51 Protein* , 1999, The Journal of Biological Chemistry.

[11]  S. Kowalczykowski,et al.  Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A , 1998, Nature.

[12]  Jeremy M. Stark,et al.  Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences , 2004, Molecular and Cellular Biology.

[13]  P. Sung,et al.  Basis for Avid Homologous DNA Strand Exchange by Human Rad51 and RPA* , 2001, The Journal of Biological Chemistry.

[14]  Y. Yoshimura,et al.  Cloning and sequence of the human RecA-like gene cDNA. , 1993, Nucleic acids research.

[15]  A. Ashworth,et al.  The BRC repeats are conserved in mammalian BRCA2 proteins. , 1997, Human molecular genetics.

[16]  S. West,et al.  CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair , 2005, Nature.

[17]  C. Larsson,et al.  Mutation analysis of the BRCA2 gene in 49 site–specific breast cancer families , 1996, Nature Genetics.

[18]  A. Stasiak,et al.  Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. , 1994, The EMBO journal.

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

[20]  G. Eichele,et al.  Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2 , 1997, Nature.

[21]  T. Ouchi,et al.  The BRCA2 gene product functionally interacts with p53 and RAD51. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A. Venkitaraman,et al.  Stabilization of stalled DNA replication forks by the BRCA2 breast cancer susceptibility protein. , 2003, Genes & development.

[23]  P. Sung.  Function of Yeast Rad52 Protein as a Mediator between Replication Protein A and the Rad51 Recombinase* , 1997, The Journal of Biological Chemistry.

[24]  C. Ingles,et al.  Interaction between BRCA2 and replication protein A is compromised by a cancer-predisposing mutation in BRCA2 , 2003, Oncogene.

[25]  Wolf-Dietrich Heyer,et al.  Human BRCA2 protein promotes RAD51 filament formation on RPA-covered ssDNA , 2010, Nature Structural &Molecular Biology.

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

[27]  S C West,et al.  Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. , 2001, Molecular cell.

[28]  N. Mazloum,et al.  DNA binding, annealing, and strand exchange activities of Brh2 protein from Ustilago maydis. , 2007, Biochemistry.

[29]  Michael G. Sehorn,et al.  Recombination Mediator and Rad51 Targeting Activities of a Human BRCA2 Polypeptide* , 2006, Journal of Biological Chemistry.

[30]  P. Bartel,et al.  RAD51 Interacts with the Evolutionarily Conserved BRC Motifs in the Human Breast Cancer Susceptibility Gene brca2 * , 1997, The Journal of Biological Chemistry.

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

[32]  Edward H Egelman,et al.  Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2 , 2007, Nature Structural &Molecular Biology.

[33]  S. Kowalczykowski,et al.  A Single-stranded DNA-binding Protein Is Needed for Efficient Presynaptic Complex Formation by the Saccharomyces cerevisiae Rad51 Protein* , 1997, The Journal of Biological Chemistry.

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

[35]  S. Kowalczykowski,et al.  Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1. , 2003, Biochemistry.

[36]  C. Dekker,et al.  Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules , 2007, Nucleic acids research.

[37]  R. McCulloch,et al.  Trypanosoma brucei BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination , 2008, Molecular microbiology.

[38]  Wen-Hwa Lee,et al.  BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure. , 2002, Science.

[39]  A. Ashworth,et al.  Mutation in Brca2 stimulates error‐prone homology‐directed repair of DNA double‐strand breaks occurring between repeated sequences , 2001, The EMBO journal.

[40]  R. Baskin,et al.  Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules , 2009, Proceedings of the National Academy of Sciences.

[41]  M. Dutreix,et al.  Real-time measurements of the nucleation, growth and dissociation of single Rad51–DNA nucleoprotein filaments , 2007, Nucleic acids research.

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

[43]  M. Jasin,et al.  BRCA2 is required for homology-directed repair of chromosomal breaks. , 2001, Molecular cell.

[44]  Ashok R. Venkitaraman,et al.  The BRC Repeats of BRCA2 Modulate the DNA-Binding Selectivity of RAD51 , 2009, Cell.

[45]  David J. Chen,et al.  Complex Formation by the Human Rad51B and Rad51C DNA Repair Proteins and Their Activities in Vitro * , 2002, The Journal of Biological Chemistry.

[46]  S. West,et al.  Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins. , 2000, Journal of molecular biology.

[47]  A. Shinohara,et al.  Stimulation by Rad52 of yeast Rad51- mediated recombination , 1998, Nature.

[48]  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.

[49]  P. Baumann,et al.  Human Rad51 Protein Promotes ATP-Dependent Homologous Pairing and Strand Transfer Reactions In Vitro , 1996, Cell.

[50]  D. Wigley,et al.  CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing. , 2006, Journal of molecular biology.

[51]  S. Kowalczykowski,et al.  DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[52]  S. Kowalczykowski,et al.  RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair. , 2003, Molecular cell.