No complementation of rad51d cells by ectopic expression of RAD51D Walker B-motif ATPase mutants Since we observed almost no abnormality in cellular pheno- type for the Walker A-motif mutations, we hypothesized that the Walker B motif may predominately determine the func- tion of RAD51D. To test this possibility, we mutated the highly conserved aspartate (D206) to alanine (D206A; see Figure 1E). For comparison purposes, we also made the pre- viously uncharacterized V203E substitution within motif B of RAD51D. This valine was chosen for mutagenesis as it is Nucleic Acids Research, 2006, Vol. 34, No. 9 2837 conserved among the human RAD51 paralogs RAD51B, RAD51C, RAD51D and XRCC3, and in mouse and hamster Rad51D. Both expression constructs (V203E and D206A) were separately transfected into rad51d cells, and cells were selected in the continuous presence of either hygromycin alone or hygromycin plus increasing concentra- tions of MMC. The extent of complementation for expression of all RAD51D constructs was assessed by comparing the number of colonies arising in hygromycin and MMC to the number of colonies arising in hygromycin alone. As shown in Figure 2C, ectopic expression of either RAD51D-V203E or RAD51D-D206A does not result in complementation of MMC sensitivity of rad51d cells measured by colony forma- tion. Conversely, in this same method of analysis, expression of wild-type RAD51D, or the Walker A box mutants RAD51D-K113R and RAD51D-K113A resulted in high MMC resistance (Figure 2C) albeit expression of either K113R or K113A did not fully protect against killing. Whether these latter modest reductions are biochemically sig- niﬁcant is unclear since the expression levels of the A-motif mutant proteins could be slightly lower than that of wild-type. As with the A-motif mutants, both B-motif mutants are expressed stably in rad51d cells (Figure 1D). Inability of RAD51D Walker B mutants to interact with XRCC2 in the yeast two-hybrid system As RAD51D B-motif mutants are unable to repair MMC- induced DNA damage, we investigated whether disruptions occur in the interactions between RAD51D and its known paralog partners. To do so, we employed the yeast two- and three-hybrid systems and tested whether mutant RAD51D would be able to interact with either XRCC2 or RAD51C. Each of the described forms of RAD51D (wild-type, K113R, K113A, V203E and D206A) was generated by site- directed mutagenesis using previously described vectors carrying wild-type RAD51D (40). First, each RAD51D protein was tested for binding to wild-type XRCC2 in both orientations (Figure 3A). The average strength of the RAD51D-K113R:XRCC2 interactions was 75 or 57% of the wild-type RAD51D:XRCC2 interaction (dependent on the orientation of the Gal4-fusion), and the average strength of Figure 2. Cell survival determined by colony formation assay after acute or chronic exposure to MMC. (A) Parental lines and high-expressing transfectants carrying Walker A-motif mutations were exposed to MMC for 60 min. AA8, solid squares; 51D1Lox (Rad51d+), open squares; 51D1 (rad51d) expressing wild-type human RAD51D (WT2), half-filled squares; 51D1 expressing Walker A-motif mutant, clone GAT4, solid diamonds; 51D1 expressing Walker A-motif mutant, clone GRT6, solid circles. (B) Parental, mutant (clone: 51D1) and low-expressing transfectants carrying Walker A-motif mutations were exposed to MMC for 60 min. Mutant 51D1 (rad51d), open diamonds; 51D1 expressing Walker A-motif mutant, clone GAT2, solid diamonds; 51D1 expre- ssing Walker A-motif mutant, clone GRT2, solid triangle; 51D1 expressing Walker A-motif mutant, clone GRT3, solid circle; for AA8, 51D1Lox and 51D1 expressing wild-type RAD51D (here clone WT1) symbols same as in (A). Horizontal line indicates D37 survival. (C) Survival of colony-forming ability in response to continuous MMC exposure of rad51d cells and transfectants expressing either wild-type RAD51D (WT; open squares), K113R (open triangles, K113A (open circles), V203E (open diamonds), D206A (closed triangles). AA8 cells (closed circles) and rad51d cells transfected with the empty vector (pcDNA3.1/hygro; black cross) are shown for comparison. Error bars that are visible are ± 1 standard deviation from three independent experiments.