2840 Nucleic Acids Research, 2006, Vol. 34, No. 9 Figure 5. Yeast two-hybrid analysis of the interactions between Walker A/B mutants of XRCC2 and RAD51D. (A) Greatly impaired XRCC2-K54A inter- action with wild-type RAD51D. Qualitative assessment: expression of b-galactosidase by X-gal filter assay; blue patches indicate the expression of interacting proteins; one representative colony is shown. (B) Pair wise analyses of the interaction between Walker A mutants from both XRCC2 and RAD51D. Qualitative assessment: as in (A). (C) Comparative analysis of the interaction between either XRCC2-L148S or XRCC2-D149A and RAD51D or between XRCC2 and RAD51D-D206A. Results are from three different colonies for every interaction, each assayed in triplicate. Error bars represent ± 1 SEM. DBD ¼ DNA binding domain of Gal4; AD ¼ transactivating domain of Gal4. newly identiﬁed Rad51 paralog in Schizosaccharomyces pombe, Rlp1, having homology to human XRCC2 shows a conserved Walker A motif but no clearly deﬁned Walker B motif (46). Therefore, we made the conservative K54R and non-conservative K54A substitutions in the Walker A motif of XRCC2, using previously described yeast two-hybrid plas- mids (40), and assayed their interaction with wild-type RAD51D, RAD51D-K113R or RAD51D-K113A qualita- tively and in both orientations by X-Gal ﬁlter assay (Figure 5A and B, respectively). XRCC2-K54A was greatly impaired in binding to wild-type RAD51D and mutant RAD51D (Figure 5A and B, respectively; light blue and white colonies). However, XRCC2-K54R was able to interact with wild-type RAD51D and both motif-A mutants of RAD51D (Figure 5A and B, respectively). In addition, we introduced L148S or D149A amino acid substitutions within the Walker B motif of XRCC2 and assessed whether these XRCC2 mutants could interact with wild-type RAD51D in the yeast two-hybrid system. Both XRCC2-L148S and XRCC2-D149A were impaired in binding to wild-type RAD51D, although B-motif mutants of XRCC2 appeared to be less detrimental to the XRCC2:RAD51D interaction than B-motif mutants of RAD51D (Figure 5C; see Discussion). DISCUSSION To our knowledge we have described the ﬁrst human ATPase with disparate requirements for two highly conserved Walker A/B motifs, both of which are generally crucial components of the nucleotide-binding site. Because of the complete loss of RAD51D function caused by amino acid substitution within its conserved B motif, the contrasting ﬁnding, of nor- mal biological function for GKT substitutions within the A motif, was quite unexpected. Moreover, a recent study of mouse Rad51D using rad51d mouse cells, in which only A-motif mutants were examined, reached the opposite con- clusion, ‘that conservation of Walker Motif A is required for a physiological role of RAD51D’ (39). Relative to the wild-type control, these authors reported 90% decrease in cell survival after exposure to MMC for K113R and K113A mutants in bulk transfected cell populations. How- ever, these investigators did not perform standard MMC dose-response measurements to calculate the dose-reduction factor of cell sensitivity associated with the mouse Rad51D mutant forms. Since we also observed a slightly increased sensitivity to MMC (calculated by dose-reduction factor) under some conditions for both acute and chronic exposures (Figure 2B and C), the only discrepancy between our survival data and theirs is the extent of reduced complementation by the A-motif mutants. In addition, Gruver et al. (39) reported moderately impaired interactions of mouse Rad51D A-motif mutants with Xrcc2, but strongly impaired interactions with Rad51C in the yeast two-hybrid system. Although we also found greatly impaired interactions between the human RAD51D Walker A mutants and RAD51C in yeast two-hybrid experi- ments when monitored qualitatively (data not shown), in the yeast three-hybrid system we found that these interactions were only moderately affected when tested in the presence of RAD51B. As discussed in the Results above, we believe that the yeast three-hybrid system more closely reﬂects the in vivo interaction context. Similar to our ﬁnding of gross defects associated with the B-motif D206A mutant of RAD51D, human XRCC3 substi- tution at the corresponding aspartate (D213N) dramatically reduced MMC resistance when expressed in hamster xrcc3 irs1SF cells (37). As suggested by others (31,37), substitution of this highly conserved aspartate likely disrupts ATP binding and/or hydrolysis activity. Nonetheless, XRCC3 does behave differently from RAD51D in that both Walker motifs are critical for XRCC3’s function (35,37). We presented evidence that alterations in the Walker B motif of RAD51D (V203E or D206A substitutions) greatly impair RAD51D-XRCC2 complex formation. Co- immunoprecipitation clearly shows that, in the presence of