2836 Nucleic Acids Research, 2006, Vol. 34, No. 9 RESULTS Complementation of rad51d cells by ectopic expression of wild-type human RAD51D Following transfection of rad51d cells [clone 51D1; (2)] with the wild-type RAD51D expression construct, colonies were selected for hygromycin resistance. To test whether these clones stably expressed the human cDNA, we picked 10 independent colonies and expanded them to mass cul- tures for western blot analysis. As shown in Figure 1A, seven of these clones stably expressed the transgene. Since the expression levels for RAD51D protein varied, one clone expressing a lower level of the protein (WT1; WT for wild- type) and one expressing a higher level (WT2) were selected to test whether ectopic expression of human RAD51D res- cued the very high ( 80-fold) MMC sensitivity of rad51d cells (2). Using the colony formation assay for cell survival, we found that both clones WT2 and WT1 were efﬁciently complemented; their MMC resistance was similar to that of the wild-type AA8 cells (Figure 2A and B, respectively). We also observed that the parent of 51D1, 51D1Lox, which contains functional Rad51D alleles prior to Cre- recombinase treatment (2), was slightly more sensitive to MMC ( 2-fold; determined from D37 values) than AA8 cells or rad51d cells expressing wild-type human RAD51D (Figure 2A and B). We explain this observation in part by the fact that the endogenous hamster Rad51D protein level in AA8 cells (and possibly the human protein in clones WT1 and WT2) is higher than in 51D1Lox cells (2). However, due to interspecies variation in antibody recognition it is not possible for us to compare the protein levels in 51D1Lox cells (i.e. hamster Rad51D) to that in the transfectants (i.e. human RAD51D). Complementation of rad51d cells by ectopic expression of RAD51D Walker A-motif ATPase mutants We tested whether a conservative amino acid substitution (K113R; binds but does not hydrolyze ATP), or a non- conservative substitution (K113A; unable to bind ATP), in the GKT box of the Walker motif A inﬂuenced the ability of RAD51D to complement the rad51d mutant. We trans- fected rad51d cells with each mutant construct (GRT and GAT; see Figure 1E) and isolated 10 independent clones for each mutant. As shown in Figure 1B and C by western blot analysis, the protein expression levels of independent clones stably expressing either mutant of RAD51D varied. As with the cells expressing the wild-type RAD51D con- struct, for each mutant we choose a low-expressing clone (GAT2 and GRT3 (GRT2 was added in follow-up experi- ments); see Figure 1C and B, respectively), and a high expressing clone (GRT6 and GAT4; see Figure 1B and C, respectively). We found that stable expression of either mut- ant protein largely complemented MMC sensitivity of rad51d cells (Figure 2A and B). The degree of MMC resistance of the high-expressing lines (GRT6 and GAT4) was similar to that of AA8 cells and rad51d cells expressing wild-type RAD51D (WT2) (Figure 2A). For the low-expressing lines (GRT2, GRT3 and GAT2), complementation was less com- plete [ 2- to 3-fold lower, as determined from the D37 values Figure 1. Representative western blots showing the expression levels for wild-type human RAD51D and its mutant forms in clonal isolates of stably transfected hamster rad51d cells. (A) rad51d clones expressing wild-type human RAD51D, Upper panel: anti-RAD51D antibody; lower panel: anti-QM antibody (loading control). Extract from untransfected rad51d cells is in left lanes of (A–D). (B) rad51d clones expressing RAD51D-K113R (GRT1-GRT6) or RAD51D-K113A (GAT5 and GAT6; loaded for compari- son); lower panel as in (A). (C) rad51d clones expressing RAD51D-K113A (GAT2-GAT5) or wild-type RAD51D (WT2; loaded for comparison purposes); lower panel as in (A). (D) rad51d clones expressing RAD51D-D206A or RAD51D-V203E. Extract of clone WT2 (expressing wild-type RAD51D) mon- itored for comparison purposes; lower panel as in (A). The asterisk indicates that V203E runs with slightly slower mobility. ‘WB’ indicates antibody used in western blot analysis. ‘WB’ indicates antibody used in western blot analysis (A–D). (E) Domain structure of the RAD51D protein illustrating the amino acid substitutions introduced into the RAD51D cDNA. (A): Walker A motif; (B): Walker B motif. Highly conserved residues are underlined in the wild-type sequence. Amino acid substitutions introduced are bold italics. Numbers in- dicate residue location within the primary sequence of RAD51D. (Figure 2B)]. However, these lower expressing lines were clearly no more sensitive to MMC than parental 51D1Lox cells that express reduced levels of hamster Rad51D [Figure 2B; (2)].