Nucleic Acids Research, 2006, Vol. 34, No. 9 2833–2843 doi:10.1093/nar/gkl366 Disparate requirements for the Walker A and B ATPase motifs of human RAD51D in homologous recombination Claudia Wiese*, John M. Hinz1, Robert S. Tebbs 1 , Peter B. Nham1, Salustra S. Urbin1, David W. Collins, Larry H. Thompson1 and David Schild Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA and 1Biosciences Directorate, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551, USA Received April 18, 2006; Accepted April 26, 2006 ABSTRACT In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the require- ment for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks (ICLs). Ectopic expression of wild-type human RAD51D or mutants having a non- functional A or B motif was used to test for comple- mentation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very effi- ciently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three- hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is neces- sary for RAD51D’s interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs. INTRODUCTION DNA double-strand breaks (DSBs) represent a severe form of DNA damage that can lead to mutations and cell death if repaired incorrectly. Cells have evolved multiple mechanisms to repair DSBs, including the high ﬁdelity process of homologous recombinational repair (HRR) (1,2), which con- tributes signiﬁcantly to the repair of DSBs in cycling cells (3), such as those generated at the replication fork during interstrand crosslink (ICL) removal. In vertebrates, HRR is highly conserved and mediated by the bacterial RecA ortholog, the Rad51 recombinase and ﬁve Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C and Rad51D). Though the biochemical functions of these paralogs in HRR are not well understood, they have been proposed to function in early steps (4–7) and in late steps (7–11) of recombination. The Rad51 paralogs are necessary for cellular resistance to DSBs (12,13), have no functional redundancy (14) and share limited sequence homology with Rad51 and with each other [ 20% identity; reviewed in (15)]. The regions of highest sequence homology are the putative ATP-binding domains: the conserved Walker A and Walker B motifs (12,13,15,16). In human cells, several RAD51 paralog complexes have been identiﬁed, among them the RAD51B-RAD51C- RAD51D-XRCC2 heterotetramer, and three heterodimers: RAD51B-RAD51C, RAD51D-XRCC2 and RAD51C-XRCC3 (4,17–24). The RAD51B-RAD51C and RAD51D-XRCC2 complexes were shown to bind to single- and double-stranded DNA and to hydrolyze ATP, an activity enhanced by DNA binding (4,19,25,26). The heterotetramer also exhibited ATPase activity and preferentially bound to Y-shaped DNA and synthetic Holliday junctions (27). The Walker ATPase domain (28) consists of two separate motifs, A and B, crucial components of the nucleotide- binding site. The lysine residue in the GKT/S box of the Walker A motif is essential for ATP-binding and contacts the g-phosphate of the nucleotide (29). The highly conserved aspartate/glutamate of the Walker B motif forms hydrogen bonds with the threonine/serine of the GKT/S box and with a bound water molecule (30) and facilitates nucleotide hydrolysis. *To whom correspondence should be addressed. Tel: +1 510 486 4024; Fax: +1 510 486 6816; Email: cwiese@lbl.gov The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org