The ATPase motif in RAD51D is required for resistance to DNA interstrand crosslinking agents and interaction with RAD51C

Homologous recombination (HR) is a mechanism for repairing DNAinterstrand crosslinks and double-strand breaks. In mammals,HR requires the activities of the RAD51 family (RAD51, RAD51B,RAD51C, RAD51D, XRCC2, XRCC3 and DMC1), each of which containsconserved ATP binding sequences (Walker Motifs A and B). RAD51Dis a DNA-stimulated ATPase that interacts directly with RAD51Cand XRCC2. To test the hypothesis that ATP binding and hydrolysisby RAD51D are required for the repair of interstrand crosslinks,site-directed mutations in Walker Motif A were generated, andcomplementation studies were performed in Rad51d-deficient mouseembryonic fibroblasts. The K113R and K113A mutants demonstrateda respective 96 and 83% decrease in repair capacity relativeto wild-type. Further examination of these mutants, by yeasttwo-hybrid analyses, revealed an 8-fold reduction in the abilityto associate with RAD51C whereas interaction with XRCC2 wasretained at a level similar to the S111T control. These cell-basedstudies are the first evidence that ATP binding and hydrolysisby RAD51D are required for efficient HR repair of DNA interstrandcrosslinks. ² Present address: Genentech, Inc., South San Francisco, CA, USA³ Present address: University of California, Davis, Sacramento,CA, USA ^* To whom correspondence should be addressed at Department of Physiology and Cardiovascular Genomics, Medical University of Ohio, Block Health Science Building, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA. Tel: +1 419 383 4370; Fax: +1 419 383 6168; Email: dpittman{at}meduohio.edu.