2838 Nucleic Acids Research, 2006, Vol. 34, No. 9 Figure 3. Yeast two- and three-hybrid analyses of the interactions of wild-type or mutant RAD51D with XRCC2 or the RAD51C:RAD51B heterodimer, respectively. (A) Quantitative assessment of the RAD51D:XRCC2 inter- action: b-galactosidase units determined photospectrometrically by using o-nitrophenyl-b-galactopyranoside as a substrate (1 U corresponds to DOD420 nm/min/mg protein). (B) Quantitative assessment of the RAD51D: RAD51B:RAD51C interaction: b-galactosidase units determined as in (A). Results in (A and B) are from three to five different colonies for every inter- action, each assayed in triplicate. Error bars represent ± 1 SEM; error bars that are not visible are too small to display. DBD ¼ DNA binding domain of Gal4; AD ¼ transactivating domain of Gal4; NF ¼ non-fused protein (here: RAD51B). (C) Western blot to monitor the expression of Gal4AD-RAD51D wild-type and mutant forms in yeast Y190U-ura transformants. Three colonies for each transformation were tested, and a representative blot is shown. Upper panel, anti-RAD51D antibody; lower panel, anti-actin antibody loading control. RAD51D-K113A:XRCC2 interactions was very close to wild-type levels (100 and 90%, depending on the orientation tested; see Figure 3A). Strikingly, both B-motif mutants displayed greatly reduced interaction with XRCC2 in either fusion orientation (Figure 3A). The average strength of the RAD51D-V203E: XRCC2 interactions was determined to be at 1 and 3% of the wild-type interaction, and the average strength of the RAD51D-D206A:XRCC2 interactions was at 3 and 11% of the wild-type interaction (depending on the orientation tested). To ensure that the RAD51D mutants are produced in amounts adequate to detect positive protein interactions in the yeast two-hybrid system, we tested the protein expres- sion levels for Gal4-RAD51D and its mutant forms by immunoblotting of total protein extracts (Figure 3C). As for the wild-type, all mutant forms of the Gal-RAD51D fusions were produced at high levels in yeast. Although K113R, V203E and D206A were repeatedly expressed at slightly lower levels than the wild-type and K113A, the failure of XRCC2 to interact with V203E and D206A is not due to the unavailability of these mutant proteins in yeast. For exam- ple, even though K113R and V203E are expressed at similar levels, they differ greatly in their ability to interact with XRCC2 (see Figure 3A and 3C). Inability of RAD51D Walker B mutants to interact with RAD51C in the yeast three-hybrid system The interaction between human RAD51D and RAD51C, when tested in the yeast two-hybrid system, is comparatively weak, making it difﬁcult to quantitatively assess protein– protein interactions that potentially are reduced even further. However, co-expression of RAD51B, in a non-fused form, has been shown previously to greatly enhance the strength of the RAD51D:RAD51C interaction in a yeast three-hybrid system (40). Furthermore, in vivo a stable RAD51D-RAD51C heterodimer has not been discovered, whereas RAD51D and RAD51C were shown to be part of the heterotetramer that also contains RAD51B and XRCC2 (20–22,24). For these reasons, we used the yeast three-hybrid system to assess the ability of either wild-type or mutant RAD51D to interact with RAD51C when RAD51B is co-expressed. We found that the average strength of interactions between Walker A K113R and RAD51C was reduced to 60% of the wild-type level, and the average strength of interactions between Walker A K113A and RAD51C was at 67% of the wild-type interac- tion (Figure 3B). Notably, the interactions between RAD51D Walker B mutants and RAD51C were virtually eliminated, not differing from the negative control (Figure 3B). No co-immunoprecipitation of RAD51D Walker B mutants with hamster Xrcc2 To investigate in vivo complex formation between wild-type or mutant human RAD51D and hamster Xrcc2, we performed reciprocal co-immunoprecipitation in extracts from rad51d clones that stably express the ectopic proteins at similar levels. We found that hamster Xrcc2 is present speciﬁcally in anti-RAD51D immunoprecipitated complexes from clone WT2 (which overexpresses wild-type RAD51D) and not immunoprecipitated using IgG control antibody (Figure 4A). In addition, CHO cells appear to express two isoforms of Xrcc2 (as indicated by the double arrows in Figure 4A–C) that both bind to RAD51D. The faster migrating isoform displays the same mobility as recombinant human XRCC2 (Figure 4C), while the second isoform appears to be larger and migrates with a shift that corresponds to a 2–3 kD difference in molecular weight. It is likely, but not clear yet, that this larger form of hamster Xrcc2 may be a modiﬁed version of the protein. We also have observed both isoforms of Xrcc2 in V79 hamster cells but not in mouse mammary epithelial cells (data not shown). Importantly, Xrcc2 was present in anti-RAD51D immunoprecipitated