blot analysis was carried out as described elsewhere (22), and ﬁlters were probed with 1:2000 diluted primary anti- RAD51D (Novus Biologicals). The signal for actin (dilution 1:300; Santa Cruz Biotech) was used as a loading control. Detection was carried out using horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Jack- son ImmunoResearch; 0.4 mg/ml). Filters were incubated with Supersignal WestPico substrate kit (Pierce Chemical) followed by exposure to Hyperﬁlm ECL (Amersham Biosciences). Expression and purification of recombinant RAD51D and XRCC2 by affinity chromatography For recombinant expression of GST-XRCC2 and His6- RAD51D, respective cDNAs were inserted into the bac- ulovirus transfer vector pFASTBAC DUAL (Invitrogen). The glutathione S-transferase (GST) ORF ﬂanked by a PreScission Protease cleavage site was inserted upstream and in frame with the XRCC2 ORF. For PCR ampliﬁcation of the RAD51D cDNA an N-terminal His6-tag was built in the forward primer. Proteins were expressed in Sf9 insect cells and puriﬁed to 80% homogeneity. One L of insect cell culture was harvested at 72 h post-infection, and cells were pelleted at 1600 g (Sorvall GSA rotor) and resuspended in 20 ml of cell lysis buffer [20 mM HEPES (pH 8.0), 100 mM NaCl, 10% glycerol, 0.5 mM phenylmethylsul- fonylﬂuoride (PMSF), 5 mg/ml aprotinin and 5 mg/ml leupeptin]. The cell suspension was incubated on ice for 20 min, sonicated and clariﬁed by ultracentrifugation at 110 000 g (Beckman 70.1 Ti rotor) for 20 min. The supernat- ant was applied to a 5 ml Glutathione Sepharose 4 FF column (Amersham Biosciences) equilibrated with 10 column volumes buffer A [1 · phosphate buffered saline (PBS), 10% glycerol]. Bound GST-XRCC2:His6-RAD51D complex was washed with 10 column volumes buffer A and eluted with buffer B [10 mM glutathione, 50 mM Tris–HCl (pH 8.0), 10% glycerol]. Peak fractions were pooled and dia- lyzed into PreScission Protease cleavage buffer [50 mM Tris– HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, 5 mg/ml aprotinin and 5 mg/ml leupeptin] for subsequent cleavage by PreScission Protease according to the manufacturer’s description (Amersham Biosciences). Following cleavage, the protein was diluted 1:1 with 100% glycerol and stored at 80 C. Transfection of rad51d cells Transfectants of rad51d cells (2) that stably express wild-type or mutant human RAD51D protein were created by electro- poration (625 V/cm, 950 mF) using Gene Pulser II (Bio-Rad). A total of 1 · 106 cells were electroporated in 400 ml cytomix buffer using 10 mg of linearized plasmid (45). Twenty-four hours post-transfection, cells were plated into 10 cm dishes at 104 cells/dish in 20 ml of medium containing hygro- mycin B (700 mg/ml) and incubated for 12 days for colony formation. Ten colonies were isolated and expanded for each transfected plasmid and analyzed for expression of the transgene by western blot analysis (22). Filters were probed with 1:2000 diluted primary anti-RAD51D (Novus Biologi- cals). The signal for the transcription factor QM (dilution Nucleic Acids Research, 2006, Vol. 34, No. 9 2835 1:10 000; Santa Cruz Biotech) was used as a loading control. Detection was carried out using HRP-conjugated goat anti-rabbit secondary antibody (Jackson Immuno- Research; 0.4 mg/ml). Filters were incubated with Super- signal WestPico substrate kit (Pierce Chemical) followed by exposure to Hyperﬁlm ECL (Amersham Biosciences). Genetic complementation analysis MMC sensitivity was determined by colony formation assay in 10 cm dishes. Exposure to MMC (Sigma) was conducted in 10 ml suspension cultures. For each dose, 1 · 106 cells were exposed to MMC for 60 min at 37 C, centrifuged and resus- pended in fresh medium for plating. For each dose, 300 or more cells were plated in triplicate. After 12 days of growth, dishes were rinsed with saline, ﬁxed with 95% ethanol and stained with Gram Crystal Violet. Colonies with >50 cells were scored. To test for complementation directly in transfected cell populations, transfections were carried out as described above. Twenty-four hours post-transfection, cells were trypsi- nized and re-seeded at 4 · 104 cells/well in 6-well plates in regular growth medium containing hygromycin B without MMC, or with MMC at various concentrations. Colonies were ﬁxed and stained after 12 days of growth. The surviving fraction of colonies was calculated as the ratio of the number of colonies in selection in MMC and hygromycin B to the number of colonies in the presence of hygromycin B only. For comparison purposes, untransfected AA8 were plated at 200 cells/well in regular growth medium lacking hygromycin B, both with and without MMC. In addition, rad51d cells were transfected with the empty vector control (pcDNA3.1/ hygro) and plated at 4 · 104 cells/well in regular growth medium containing hygromycin B without or with MMC. The transfection efﬁciencies for all plasmids used were deter- mined in a separate experiment. Plasmids were electroporated as described above and cells were plated 24 h post transfec- tion in regular growth medium containing hygromycin B. The stable transfection efﬁciencies were found to be similar for all plasmids used and were 0.27 ± 0.03%. Immunoprecipitation Immunoprecipitation was performed as described previously (22) using native cellular protein extracts from untransfected rad51d and AA8 cells and rad51d cells stably expressing either wild-type or mutant RAD51D. Immunoprecipitation was carried out using MagnaBind goat anti-rabbit IgG mag- netic beads (Pierce) and anti-RAD51D antibody (Novus Bio- logicals) or control antibody (rabbit IgG, Santa Cruz Biotech). The polyclonal rabbit anti-human XRCC2 antibody for direct immunoprecipitation of hamster Xrcc2 was kindly provided by Dr P. Sung. Western blots were probed with 1:2000 diluted primary anti-RAD51D and 1:1000 diluted primary anti-XRCC2 antibody (Santa Cruz Biotech). Detec- tion was carried out using HRP-conjugated goat anti-rabbit secondary antibody (see above) or bovine anti-goat secondary antibody (1:10 000; Santa Cruz Biotech). Filters were incu- bated as described above for analysis of RAD51D-expressing rad51d cells. To visualize co-immunoprecipitated hamster Xrcc2, ﬁlters were incubated with ECL-Advance (Amersham Biosciences).