Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions.

The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2-mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms.

[1]  M. Wold,et al.  Recombinant replication protein A: expression, complex formation, and functional characterization. , 1994, The Journal of biological chemistry.

[2]  Shigeyuki Yokoyama,et al.  Crystal structure of the homologous-pairing domain from the human Rad52 recombinase in the undecameric form. , 2002, Molecular cell.

[3]  A. Ashworth,et al.  Structural analysis of the chicken BRCA2 gene facilitates identification of functional domains and disease causing mutations. , 2002, Human molecular genetics.

[4]  Janice P. Evans,et al.  BRCA2 deficiency in mice leads to meiotic impairment and infertility , 2004, Development.

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

[6]  S. Boulton,et al.  RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair , 2005, Molecular and Cellular Biology.

[7]  T. Blundell,et al.  Dynamic control of Rad51 recombinase by self-association and interaction with BRCA2. , 2003, Molecular cell.

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

[9]  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.

[10]  Tom L. Blundell,et al.  Insights into DNA recombination from the structure of a RAD51–BRCA2 complex , 2002, Nature.

[11]  W. Holloman,et al.  Brh2-Dss1 Interplay Enables Properly Controlled Recombination in Ustilago maydis , 2005, Molecular and Cellular Biology.

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

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

[14]  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.

[15]  Ashok R Venkitaraman,et al.  Cancer Susceptibility and the Functions of BRCA1 and BRCA2 , 2002, Cell.

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

[17]  M. Jasin,et al.  Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. , 1998, Genes & development.

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

[19]  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.

[20]  M. Wold.  Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. , 1997, Annual review of biochemistry.

[21]  M. Moynahan,et al.  The cancer connection: BRCA1 and BRCA2 tumor suppression in mice and humans , 2002, Oncogene.

[22]  D. Ward,et al.  Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[23]  S. West,et al.  BRCA2 BRC motifs bind RAD51-DNA filaments. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[24]  F. Gage,et al.  Truncated BRCA2 is cytoplasmic: implications for cancer-linked mutations. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[25]  A. Buchman,et al.  BRCA2 homolog required for proficiency in DNA repair, recombination, and genome stability in Ustilago maydis. , 2002, Molecular cell.

[26]  R. Wooster,et al.  Breast and ovarian cancer. , 2003, The New England journal of medicine.

[27]  S. Seal,et al.  Biallelic BRCA2 mutations are associated with multiple malignancies in childhood including familial Wilms tumour , 2005, Journal of Medical Genetics.

[28]  S. West,et al.  Structure of the single-strand annealing domain of human RAD52 protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[29]  M. Jasin.  Homologous repair of DNA damage and tumorigenesis:the BRCA connection , 2002, Oncogene.

[30]  R. Kanaar,et al.  Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions , 2002, Molecular and Cellular Biology.

[31]  Zhao-Qi Wang,et al.  Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[34]  M. Doutriaux,et al.  Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1 , 2004, The EMBO journal.

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

[36]  Jakub Tolar,et al.  Germline mutations in BRCA2: shared genetic susceptibility to breast cancer, early onset leukemia, and Fanconi anemia. , 2004, Blood.

[37]  E. Bochkareva,et al.  From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold. , 2004, Current opinion in structural biology.

[38]  J. Haber,et al.  Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae , 1999, Microbiology and Molecular Biology Reviews.

[39]  Jeremy M. Stark,et al.  ATP Hydrolysis by Mammalian RAD51 Has a Key Role during Homology-directed DNA Repair* , 2002, The Journal of Biological Chemistry.

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

[41]  N. Ellis,et al.  Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. , 2001, Genes & development.

[42]  Kenneth Offit,et al.  Shared genetic susceptibility to breast cancer, brain tumors, and Fanconi anemia. , 2003, Journal of the National Cancer Institute.

[43]  Sara K. Binz,et al.  Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface. , 2003, Biochemistry.

[44]  Michael G. Sehorn,et al.  Rad51 Recombinase and Recombination Mediators* , 2003, Journal of Biological Chemistry.