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 DNA interstrand 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 contains conserved ATP binding sequences (Walker Motifs A and B). RAD51D is a DNA-stimulated ATPase that interacts directly with RAD51C and XRCC2. To test the hypothesis that ATP binding and hydrolysis by RAD51D are required for the repair of interstrand crosslinks, site-directed mutations in Walker Motif A were generated, and complementation studies were performed in Rad51d-deficient mouse embryonic fibroblasts. The K113R and K113A mutants demonstrated a respective 96 and 83% decrease in repair capacity relative to wild-type. Further examination of these mutants, by yeast two-hybrid analyses, revealed an 8-fold reduction in the ability to associate with RAD51C whereas interaction with XRCC2 was retained at a level similar to the S111T control. These cell-based studies are the first evidence that ATP binding and hydrolysis by RAD51D are required for efficient HR repair of DNA interstrand crosslinks.
Gruver
Aaron M
AM
Department of Physiology and Cardiovascular Genomics, Medical University of Ohio, Toledo, OH 43614-5804, USA.
Miller
Kristi A
KA
Rajesh
Changanamkandath
C
Smiraldo
Phillip G
PG
Kaliyaperumal
Saravanan
S
Balder
Rachel
R
Stiles
Katie M
KM
Albala
Joanna S
JS
Pittman
Douglas L
DL
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D012333:RNA, Messenger; C491807:Rad51d protein, mouse; C491866:Xrcc2 protein, mouse; D016685:Mitomycin; D004247:DNA; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases