XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

Journal Article · Wed Feb 25 04:00:00 EST 2004 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M402247200· OSTI ID:15014151

Yamada, N; Hinz, J; Kopf, V L; Segalle, K; Thompson, L

Homologous recombinational repair is a major DNA repair pathway that preserves chromosomal integrity by removing double-strand breaks, crosslinks, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 appears to exist in a single complex with Rad51C. To begin 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 non-conservative 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 CHO cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, while 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 the mutants' dysfunction, 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 coexpression in bacteria, nickel affinity purification, and western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, while the K113R mutant did not and was predominantly insoluble. Addition of 5 mM ATP, but not ADP, also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 is likely to regulate the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis, with both processes being essential for the complex's ability to participate in HRR.

• Research Organization: Lawrence Livermore National Laboratory (LLNL), Livermore, CA
• Sponsoring Organization: USDOE
• DOE Contract Number: W-7405-ENG-48
• OSTI ID: 15014151
• Report Number(s): UCRL-JRNL-203718
• Journal Information: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 22 Vol. 279; ISSN 0021-9258; ISSN JBCHA3
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
CHO CELLS
DNA DAMAGES
DNA REPAIR
IN VITRO
RECOMBINATION