TY - JOUR
T1 - The ATPase motif in RAD51D is required for resistance to DNA interstrand crosslinking agents and interaction with RAD51C
AU - Gruver, Aaron M.
AU - Miller, Kristi A.
AU - Rajesh, Changanamkandath
AU - Smiraldo, Phillip G.
AU - Kaliyaperumal, Saravanan
AU - Balder, Rachel
AU - Stiles, Katie M.
AU - Albala, Joanna S.
AU - Pittman, Douglas L.
N1 - Funding Information:
We thank Kandace Williams, Jean Overmeyer and Aditi Nadkarni for critical reading of the manuscript. This work was supported by an American Cancer Society grant to D.L.P. (RSG-030158-01-GMC). A.M.G. was supported by an MD/PhD predoctoral fellowship from the Medical University of Ohio. Financial support was given by The American Cancer Society.
PY - 2005/11
Y1 - 2005/11
N2 - 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.
AB - 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.
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U2 - 10.1093/mutage/gei059
DO - 10.1093/mutage/gei059
M3 - Article
C2 - 16236763
AN - SCOPUS:29144533468
SN - 0267-8357
VL - 20
SP - 433
EP - 440
JO - Mutagenesis
JF - Mutagenesis
IS - 6
ER -