these non-complementing RAD51D mutant proteins, there is much less hamster Xrcc2 protein than under conditions where complementing RAD51D (i.e. wild-type or motif-A mutants) is available. It is important to note from these experiments that the rad51d knockout cell line itself (i.e. untransfected) also expresses greatly reduced levels of endogenous Xrcc2 protein when compared to its AA8 parent line, which expresses wild- type hamster Rad51D (Figure 4C). Our ﬁndings are consistent with several studies showing the stabilization of RAD51 paralogs by each other in vivo (7,22,47,48), but our data addi- tionally demonstrate that the ability to interact directly is required for this stabilization. In addition, neither Walker B-motif mutant is able to interact with the RAD51B:RAD51C heterodimer in the yeast three-hybrid system. The loss of RAD51D-XRCC2 and RAD51D-RAD51C interactions by disruption of nucleotide binding (V203E or D206A substitutions) raises the question of which biochemical defect (nucleotide binding or protein–protein interaction) is responsible for the cellular phenotype of high sensitivity to MMC. Our preferred view is that the role of the ATPase activ- ity that is blocked by the V203E and D206A substitutions is to mediate a conformational change that promotes tight binding of RAD51D with XRCC2 and/or RAD51C. In this model ATPase site integrity and protein interaction are inseparable. Reiterative changes in protein conformation are important for the assembly of multi-subunit protein complexes that function in DNA repair [for review see (49)]. Rad51 family members induce these conformational changes, necessary to fulﬁll protein function, by binding and hydrolysis of ATP [for review see (30)]. X-ray crystal structures (50–54) and electron microscopy reconstructions (53–55) of several Rad51 homologs have provided evidence that ATP-binding occurs at the subunit interface by contacting several highly conserved residues on neighboring subunits. We surmise that human RAD51D, through its Walker B motif, may rely on the A motif of another RAD51 paralog to form an active ATPase. Overall, our results suggest that RAD51D must form a complex with XRCC2 and/or RAD51C to maintain cellular resistance to MMC. We showed that both interactions require a normal RAD51D Walker B motif, but not an intact A motif. We speculate that the binding of ATP to RAD51D occurs through its Walker B box as part of a bipartite nucleotide- binding site interacting with XRCC2 and/or RAD51C, as illustrated in Figure 6. In Figure 6A, the B motif of RAD51D interacts with the A motif of XRCC2. We showed that mutations in either of these motifs block the interaction; disruption of the XRCC2 A motif via K54A substitution (but not K54R substitution) greatly diminishes its binding to RAD51D in the yeast two-hybrid system (Figure 5A and B). One published observation is inconsistent with the idea that the RAD51D-XRCC2 interaction is important for efﬁci- ent DNA repair. In that study, the XRCC2 A-motif K45A mutant showed only modest decrements in MMC resistance in irs1 V79 cells transfected with mutant proteins (36) (although western blotting of these transfectants was not used to standardize the expressed XRCC2 protein levels). Moreover, in our yeast two-hybrid studies amino acid substi- tutions within the Walker B motif of XRCC2, though clearly affecting the XRCC2:RAD51D interaction, appear to be less deleterious to XRCC2:RAD51D complex formation in Nucleic Acids Research, 2006, Vol. 34, No. 9 2841 Figure 6. Possible configurations of RAD51D interactions involving bipartite Walker A and B nucleotide binding motifs. (A) The RAD51D-XRCC2 hetero- dimer requires both the B motif of RAD51D and the A motif of XRCC2. (B) The RAD51D-RAD51C heterodimer requires the B motif of RAD51D; a require- ment for the A motif of RAD51C is predicted but not yet been tested. Neither heterodimer requires the A motif of RAD51D. The possible role of the B motif of RAD51C has not been tested. comparison with the RAD51D B-motif mutants (Figure 5C). Figure 6B depicts an ATPase site composed of the B motif of RAD51D and the A motif of RAD51C. Similar to our results for the B motif of RAD51D, mutations in the A motif of RAD51C also cause 80–100% loss of DNA repair efﬁciency (34). Although RAD51D-RAD51C interactions were not examined in that study, we found a severe RAD51- D:RAD51C interaction defect in the yeast three-hybrid system for the RAD51D B-motif mutants (Figure 3). In conclusion, we provide evidence that the Walker B motif of human RAD51D is critically important for com- plex formation with other RAD51 paralogs and for cellular resistance to ICLs. Since both Walker B-motif substitutions (V203E and D206A) yielded a similar phenotype, we propose that these substitutions not only abrogate ATP hydrolysis, but also simultaneously prevent one or more critical interac- tion(s) and conformational changes underlying RAD51D’s participation in complexes, whose integrity is necessary for recombinational repair. ACKNOWLEDGEMENTS The authors are grateful to Drs M.-S. Tsai and M. Berardini for baculovirus transfer vector construction and help with protein purification. This work was supported by NCI/NIH P01 grant CA92584 (D.S.) titled Structural Cell Biology of DNA Repair Machines and NASA grant NNJ05HI36I (C.W.). The portion of this work done at LLNL was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48 where the DOE Low-Dose Program and NCI/ NIH grants CA89405 and CA112566 funded this work. Funding to pay the Open Access publication charges for this article was provided by NCI/NIH P01 grant CA92584. Conflict of interest statement. None declared. REFERENCES 1. Helleday,T. (2003) Pathways for mitotic homologous recombination in mammalian cells. Mutat. Res., 532, 103–115. 2. Hinz,J.M., Tebbs,R.S., Wilson,P.F., Nham,P.B., Salazar,E.P., Nagasawa,H., Urbin,S.S., Bedford,J.S. and Thompson,L.H. (2006) Repression of mutagenesis by Rad51D-mediated homologous recombination. Nucleic Acids Res., 34, 1358–1368.