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M402247200v1
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Papers In Press, published online ahead of print March 22, 2004
J. Biol. Chem, 10.1074/jbc.M402247200
Submitted on March 1, 2004
Revised on March 22, 2004
Accepted on March 22, 2004

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

Nazumi A. Yamada, John M. Hinz, Vicki L. Kopf, Kathryn D. Segalle, and Larry H. Thompson

Biology & Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551-0808

Corresponding Author: thompson14{at}llnl.gov

Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, crosslinks, 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 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 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, 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.


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