title: XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination.
abstract: Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, cross-links, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2/3, Rad51B/C/D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 exists in a single complex with Rad51C. To examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a nonconservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF Chinese hamster ovary cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, whereas ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of dysfunction of the mutants, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon co-expression in bacteria, nickel-affinity purification, and Western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, whereas the K113R mutant did not and was predominantly insoluble. The addition of 5 mm ATP but not ADP also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 probably regulates the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis with both processes being essential for the ability of the complex to participate in homologous recombinational repair.
authors.affiliation: Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551-0808, USA.
authors.forename: Nazumi Alice
authors.initials: NA
authors.lastname: Yamada
authors.forename: John M
authors.initials: JM
authors.lastname: Hinz
authors.forename: Vicki L
authors.initials: VL
authors.lastname: Kopf
authors.forename: Kathryn D
authors.initials: KD
authors.lastname: Segalle
authors.forename: Larry H
authors.initials: LH
authors.lastname: Thompson
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D006224:Cricetinae; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D006801:Humans; D006868:Hydrolysis; D011485:Protein Binding; D011995:Recombination, Genetic
chemical_list: D004268:DNA-Binding Proteins; C448305:RAD51C protein, human; C110901:X-ray repair cross complementing protein 3; D000251:Adenosine Triphosphatases