Nucleic Acids Research, 2006, Vol. 34, No. 9 2839 Figure 4. RAD51D wild-type and motif-A mutants allow complex formation with hamster Xrcc2 in vivo. (A) Crude extract from clone WT2 (overexpressing wild- type human RAD51D) immunoprecipitated with a polyclonal antibody against RAD51D. Precipitated RAD51D (upper panel) and co-precipitated Xrcc2 (lower panel) were detected by western blot analysis on the same membrane. A mock immunoprecipitate by incubation of WT2 extracts with IgG control antibody is shown in the far left lane. For comparison purposes, whole cell extract from clone WT2 is shown in the far right lane. Note: Xrcc2 is present in two isoforms in CHO cells [strain AA8 and derivatives; see also (C)]. ‘HC’ indicates cross-reacting heavy chain signal. ‘IP’ indicates antibody used for immunoprecipitation. ‘WB’ indicates antibody used in western blot. (B) Presence of Xrcc2 in anti-RAD51D immunoprecipitated complexes from clones WT2 (GKT), GRT6 (K113R) and GAT4 (K113A). Xrcc2 is not present in anti-RAD51D complexes from parental AA8 or rad51d cells (neither cell line expresses human RAD51D that is precipitated here). For comparison, crude extracts from human cells (HeLa; far left lane), WT2 cells (GKT; second lane from the right), and AA8 cells (far right lane) are shown. Upper and lower panels: western blot probed with Rad51D and XRCC2 antibodies, respectively. (C) Endogenous hamster Xrcc2 is expressed at higher levels in cell lines expressing complementing RAD51D (GKT, K113R, K113A) that is able to interact with Xrcc2. Upper panel: western blot probed with anti-XRCC2 antibody shows two Xrcc2 isoforms in Xrcc2 immunoprecipitated complexes from rad51d, K113R, K113A, D206A, V203E and GKT cell lines. Precipitated Xrcc2 from AA8 cells is shown for comparison (far left lane; note that only 1/16 of the precipitated complex relative to all other cell lines was loaded in order to avoid a very strong signal for Xrcc2). Two different concentrations of recombinant human XRCC2/(His)6-RAD51D complex isolated from insect cells (second and third lanes from the left) and crude extract from clone WT2 (GKT; far right lane) are included. Lower panel: same blot probed with RAD51D antibody to detect human RAD51D that co- immunoprecipitated with hamster Xrcc2. Note: recombinant human His6-tagged RAD51D migrated more slowly than human RAD51D. complexes from rad51d cells overexpressing A-motif mutants (K113R and K113A), but absent in anti-RAD51D immunoprecipitated complexes from B-motif mutants (D206A and V203E; Figure 4B). Although reproducibly at lower levels compared to the wild-type expressing line (WT2; here: GKT), Xrcc2 was always present in anti- RAD51D immunoprecipitated complexes from clones GAT4 and GRT6, which overexpress Walker A K113A and K113R mutant protein, respectively. Anti-RAD51D immuno- precipitated complexes from parental AA8 and rad51d cells contained neither RAD51D nor Xrcc2, demonstrating the inability of the anti-RAD51D antibody to recognize the ham- ster Rad51D protein in AA8 cells and the absence of human RAD51D in rad51d cells. In reciprocal experiments we analyzed anti-Xrcc2 immunoprecipitated complexes from all cell lines for the presence of RAD51D (Figure 4C). We found that the amounts of endogenous Xrcc2 directly precipitated by anti- XRCC2 antibody varied greatly among all cell lines tested (even though their expression levels for ectopic RAD51D were comparable; see Figure 1). Only small amounts of Xrcc2 were precipitated from extracts of rad51d cells and rad51d cells expressing Walker B-motif mutants D206A or V203E (Figure 4C). Signiﬁcantly larger amounts of Xrcc2 were precipitated from cells overexpressing wild-type RAD51D, K113R or K113A and from AA8 cells. (Note: for AA8 cells only 1/16 of the amount of the precipi- tated complex was loaded onto the gel, relative to all other cell lines). We reproducibly detected wild-type RAD51D (here: GKT), K113R and K113A in anti-Xrcc2 immunopre- cipitated complexes (Figure 4C, lower panel). Not surpris- ingly, only small amounts of Walker B mutant proteins were present in anti-Xrcc2 complexes from rad51d cells overexpressing either V203E or D206A (Figure 4C). In sum, these results show that ectopically expressed wild-type RAD51D, or RAD51D with mutations in the Walker A motif, formed a stable complex with hamster Xrcc2. Conversely, motif-B mutants, although expressed at similar levels, had reduced levels of hamster Xrcc2, probably due to the inability to stabilize this protein as a RAD51D-Xrcc2 heterodimer (Figure 5C; see Discussion). Greatly impaired interaction of XRCC2-K54A and RAD51D in the yeast two-hybrid system As we observed little decrease in interaction between RAD51D A-motif mutants and Xrcc2/XRCC2 both in vivo and in the yeast two-hybrid system, we hypothesized that XRCC2’s Walker A motif participates in nucleotide-binding within the XRCC2-RAD51D heterodimer. Interestingly, a