2834 Nucleic Acids Research, 2006, Vol. 34, No. 9 In human RAD51, non-conservative (no ATP binding) K133A substitution in the Walker A motif did not rescue the Rad51-deﬁcient lethality of chicken DT40 cells while the conservative (ATP binding, but no hydrolysis) K133R substitution only gave a partially defective phenotype (31). In in vitro assays, human RAD51-K133R showed an increased stability of presynaptic ﬁlament formation and enhanced homologous pairing activity compared to the wild-type protein (32), but in a dominant-negative approach, expression of human RAD51-K133R in a mouse embryonic stem cell line speciﬁcally inhibited homology-directed DSB repair (33). In the Walker B motif of human RAD51, muta- tion in the highly conserved aspartate (D222A), but not V221A or S223A, was shown to eliminate protein function in the DT40 system (31). For the human RAD51 paralogs, mutations within the Walker A motif of RAD51C and XRCC3 were shown to greatly reduce cellular resistance to ICLs (34,35), but mutation in the Walker A motif in XRCC2 had little effect on complementing ability (36). Sub- stitution of the conserved aspartate residue in the Walker B motif of human XRCC3 (D213N) resulted in increased spon- taneous apoptosis and complete loss of gene-complementing activity for cell survival to ICL damage in the CHO xrcc3 mutant (37,38). Evidence suggests that nucleotide binding and hydrolysis play important roles in the formation of RAD51 paralog complexes. For example, in XRCC3, the inability to hydrolyze bound ATP (i.e. a Walker A GRT sub- stitution) blocks the interaction with RAD51C (35). A study of the paralog interactions of mouse Rad51D Walker A mutants has also been reported (39), and these results are discussed later. In this study, Walker-motif mutants of RAD51D were tested for their ability to complement the MMC sensitivity of CHO rad51d knockout cells [hereafter written as rad51d cells; (2)]. Surprisingly, RAD51D mutants of the GKT box of the Walker A motif rescued the MMC sensitivity of RAD51D cells, whereas B-motif mutants did not comple- ment. In addition, whereas A-motif mutations had little effect on the interaction of rad51d with binding partners XRCC2 and RAD51C, mutations in the B-motif disrupted these inter- actions. These results constitute the novel ﬁnding of a differ- ential requirement for the Walker A and B motifs of a human ATPase. MATERIALS AND METHODS Cell culture Parental CHO cells (strain AA8) and 51D1Lox and 51D1 (rad51d) derivatives (2) were grown in monolayer or suspen- sion cultures in MEM-alpha medium supplemented with 10% fetal bovine serum, 100 mg/ml streptomycin and 100 U/ml penicillin. For the experiments involving chronic MMC expo- sures and the generation of denatured and crude protein lysates from rad51d derivatives, cells were grown under selection in 700 mg/ml hygromycin B. Human RAD51D expression plasmids The RAD51D cDNA was PCR ampliﬁed from plasmid pDS200 (40) and subcloned into pcDNA3.1/hygro (Invitrogen) from HindIII to NheI. The entire RAD51D open reading frame (ORF) was veriﬁed by direct sequenc- ing. Mutations were introduced using the QuickChange Site-directed mutagenesis II kit (Stratagene), and missense mutations were conﬁrmed by direct sequencing of the entire ORF. Mutagenic primers were designed to create the following changes to the amino acid sequence of RAD51D: K113R, 50-GGCCCAGGTAGCGGCCGC- ACTCAGGTATGTCTCTGTATGGC-30; K113A, 50-GG- CCCAGGTAGCGGCGCTACTCAGGTATGTCTCTGTAT- GGC-30; V203E, 50-GGAACTGTGAAGGTGGAGGTTG- TGGACTCGGTCACTGCGG-30; and D206A, 50-GGAACT- GTGAAGGTGGTGGTTGTGGCTTCGGTCACTGCGG-30 (only forward primers are shown; reverse primers had the complementary sequence). Yeast two- and three-hybrid system The yeast strain Y190-ura (40) was used for all yeast two- and three-hybrid experiments. For yeast two-hybrid analyses, Y190-ura was co-transformed with both a DNA-binding domain [pGBT9; (41)] and a transcription-activating domain fusion plasmid [pGAD424; (41)] and transformants were recovered on selective media (i.e. synthetic complete media lacking leucine and tryptophan: SC Leu, Trp). For yeast three-hybrid analyses, RAD51B was expressed from pVT100U, a yeast expression vector containing the URA3 selectable marker (42) as described in (40). To test three- hybrid interactions, strains containing two plasmids, grown in SC Leu, Ura, were transformed with the third plasmid, derived from pGBT9, and selected on plates lacking leucine, tryptophan and uracil. Four independent colonies for all trans- formations were assayed qualitatively for b-galactosidase activity using X-gal ﬁlter assay (43). To quantify the strength of protein–protein interactions, liquid b-galactosidase assays were performed using o-nitrophenyl-b-galactopyranoside (Sigma) as a substrate as described (43). All quantitative b-galactosidase activity assays were performed on three to ﬁve independent colonies, each monitored in triplicate. Stan- dard error of the mean (SEM) was determined among inde- pendent colonies. Yeast two-hybrid plasmids for RAD51D, XRCC2 and RAD51C are described in (40). Mutations were introduced into the RAD51D using site-directed mutagenesis and mutagenic primers as described above. Mutations were introduced into the XRCC2 ORF using the QuickChange site-directed mutagenesis II kit (Stratagene). Missense mutations were conﬁrmed by direct sequencing of the entire ORF. Mutagenic primers for XRCC2 were designed as follows: K54R, 50-GGCCCAGAAGGAACAGGACGCAC- AGAAATGC-30; K54A, 50-GGCCCAGAAGGAACAGGA- GCTACAGAAATGC-30; D149A, 50-GCCTTTTGATTT- TGGCCAGCCTGTCAGCTTTTTACTGG-30; L148S, 50-GC- CTTTTGATTTCAGATAGCCTGTCAGCTTTTTACTGG-30; (only forward primers are shown; reverse primers had the complementary sequence). Whole cell protein extracts from transformed yeast strain Y190U-ura To monitor the expression levels of Gal4-RAD51D in Y190U-ura transformants, whole cell protein extracts were generated according to published procedures (44). Western