title: Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression.
abstract: The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.
authors.affiliation: Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1 Canada.
authors.forename: Shauna A
authors.initials: SA
authors.lastname: Lee
authors.forename: Céline
authors.initials: C
authors.lastname: Roques
authors.forename: Alissa C
authors.initials: AC
authors.lastname: Magwood
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Masson
authors.forename: Mark D
authors.initials: MD
authors.lastname: Baker
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D024682:BRCA2 Protein / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D015153:Blotting, Western; D002460:Cell Line; D004249:DNA Damage; D004265:DNA Helicases; D005786:Gene Expression Regulation; D018390:Gene Targeting; D006825:Hybridomas; D051379:Mice; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism*; D051721:Rad52 DNA Repair and Recombination Protein / Q000378:metabolism; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D024682:BRCA2 Protein; C106711:BRCA2 protein, mouse; D009687:Nuclear Proteins; D012333:RNA, Messenger; D051721:Rad52 DNA Repair and Recombination Protein; C497196:Rad52 protein, mouse; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C103534:Rad54l protein, mouse