In yeast, the Rad51-related proteins include Rad55 and Rad57, which form a heterodimer that interacts with Rad51. Five human Rad51 paralogs have been identified (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/ Rad51L2, and Rad51D/Rad51L3), and each interacts with one or more of the others. Previously we reported that HsRad51 interacts with XRCC3, and Rad51C interacts with XRCC3, Rad51B, and HsRad51. Here we report that in the yeast two-hybrid system, Rad51D interacts with XRCC2 and Rad51C. No other interactions, including self-interactions, were found, indicating that the observed interactions are specific. The yeast Rad51 interacts with human Rad51 and XRCC3, suggesting Rad51 conservation since the human yeast divergence. Data from yeast three-hybrid experiments indicate that a number of the pairs of interactions between human Rad51 paralogs can occur simultaneously. For example, Rad51B expression enhances the binding of Rad51C to XRCC3 and to HsRad51D, and Rad51C expression allows the indirect interaction of Rad51B with Rad51D. Experiments using 6xHis-tagged proteins in the baculovirus system confirm several of our yeast results, including Rad51B interaction with Rad51D only when Rad51C is simultaneously expressed and Rad51C interaction with XRCC2 only when Rad51D is present. These results suggest that these proteins may participate in one complex or multiple smaller ones.The Rad51 protein is a functional homolog of the bacterial RecA protein and is the major strand transfer protein in eucaryotic cells (1-3). In addition to Rad51, the yeast Saccharomyces cerevisiae has two proteins, Rad55 and Rad57, that share limited amino acid sequence homology with Rad51. These proteins appear to be Rad51 paralogs, probably derived by duplication of the ancestral gene encoding Rad51 but now divergent in function. The Rad55 and Rad57 proteins interact and form a tight dimer that weakly interacts with Rad51 and assists it in strand transfer, probably by helping Rad51 displace RPA from single-stranded DNA (4).Human cells have a true Rad51 homolog (HsRad51), and five mitotically expressed Rad51 paralogs have recently been identified (XRCC2, XRCC3, Rad51B/Rad51L1/HsRec2, Rad51C/ Rad51L2, and Rad51D/Rad51L3) (5-11). Like the yeast Rad51 paralogs, these proteins share limited (ϳ20 -30%) amino acid sequence homology with HsRad51 and with each other. The human XRCC2 and XRCC3 genes were isolated (5, 6, 12) and shown to complement the DNA repair defect and chromosome instability of the irs1 and irs1SF hamster-derived cell lines (13-16). Each of the paralogs has recently been knocked out in the chicken B lymphocyte line DT40, and all of the knockouts are sensitive to DNA damage and show great chromosome instability (17). 1Several lines of evidence suggest that the human Rad51 paralogs play an important, but not crucial, role in recombination. Evidence from mammalian cells indicates that the repair of DNA cross-links requires recombination (18,19). The extreme sensitivity to DNA cross-linking reagents, such as mitomycin C and cis-platin, of mamm...

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/ Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His) 6 , and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B⅐Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single-and double-stranded DNA and to preferentially bind 3-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway. Homologous recombinational repair (HRR)1 is an important pathway in repairing DNA double-strand breaks (DSBs) with high accuracy, which is indispensable for the maintenance of genome stability (for review see Refs. 1-4). The human Rad51 protein, a structural and functional homolog of Escherichia coli recombinase RecA (5, 6), plays a central role in HRR. In vitro studies have shown that Rad51 binds single-stranded and double-stranded DNA (ssDNA and dsDNA), exhibits DNA-dependent ATPase activity, and functions to catalyze homologous pairing and DNA strand exchange (6 -8). As a prerequisite step for the action of Rad51 in HRR, Rad51 binds DNA to form highly ordered nucleoprotein filaments (9, 10). It has also been shown that these filaments form preferentially on tailed duplex DNA substrates that mimic the 3Ј-overhanging ssDNA tails at the sites of DSBs (11). Thus far, Rad51 is the only mitotic protein that catalyzes the key reactions of homologous pairing and strand transfer.In yeast, the Rad51-related proteins include Rad55 and Rad57, which exist together as a tight heterodimer that interacts weakly with Rad51 (12). These two proteins appear to be Rad51 paralogs, probably derived by gene duplication followed by the evolution of new functions. The Rad55⅐Rad57 complex acts as a cofactor for the assembly of Rad51 onto ssDNA and functions to promote the DNA strand transfer activity of Rad51 (12). In human cells, in addition to meiotic Dmc1 protein, five Rad51 paralogs have been identified: Rad51B/Rad51L1 2 (13-15), Rad51C/Rad51L2 (16), Rad51D/Rad51L3 (15, 17, 18), XRCC2 (19 -21), and XRCC3 (19,22,23). These Rad51 paralogs share 20 -30% sequence id...

The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) are expressed in mitotically growing cells and are thought to play mediating roles in homologous recombination, although their precise functions remain unclear. Among the five paralogs, Rad51C was found to be a central component present in two complexes, Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2. We have shown previously that the human Rad51C protein exhibits three biochemical activities, including DNA binding, ATPase, and DNA duplex separation. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA-cross-linking agent mitomycin C and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G 2 /M phases of the cell cycle but not in G 1 phase. Together, these results provide direct cellular evidence for the function of human Rad51C in homologous recombinational repair.In mammalian cells, DNA double strand breaks (DSBs) 1 are repaired primarily by two distinct mechanisms, non-homologous end joining (NHEJ), a non-templated, potentially errorprone process in which nucleotide alternations are tolerated at the site of rejoining, and homologous recombination (HR), a largely error-free process in which a sister chromatid or homologous chromosome is used as a template for repair (for review, see Refs. 1 and 2). Homologous recombinational repair (HRR) provides high fidelity in repairing DNA damage and is therefore essential and critical for the maintenance of genome stability and tumor avoidance (for review, see Refs. 3 and 4). The Rad51 protein plays a key role in HR, functioning to mediate homologous DNA pairing and strand exchange (5, 6). Five vertebrate Rad51 paralogs are expressed in mitotically growing cells: Rad51B (7-9), Rad51C (10), Rad51D (9, 11, 12), XRCC2 (13-15), and XRCC3 (13,16,17). These proteins share 20 -30% sequence identity with Rad51 and with each other. Only vertebrates appear to contain all five of these Rad51 paralogs. In human cells, Rad51C participates in various paralog complexes, including Rad51B-Rad51C, Rad51C-XRCC3, and Rad51B-Rad51C-Rad51D-XRCC2 (18 -22). In terms of proteinprotein interactions, Rad51C apparently has a central role, interacting directly with Rad51B, Rad51D, and XRCC3 and also weakly with Rad51 (23,24). However, the functional significance of these complexes is not yet clear.Mutant studies provide a direct means for identifying the function of genes. A knock-out ...

The bystander effect for sister chromatid exchanges (SCEs) and chromosomal aberrations was examined in hamster cell lines deficient in either DNA-PKcs (V3 cells, deficient in nonhomologous end joining, NHEJ) or RAD51C (irs3 cells, deficient in homologous recombination, HR). Cells synchronized in G0/G1 phase were irradiated with very low fluences of alpha particles such that < 1% of the nuclei were traversed by an alpha particle. Wild-type cells showed a prominent bystander response for SCE induction; an even greater effect was observed in V3 cells. On the other hand, no significant induction of SCE was observed in the irs3 RAD51C-deficient bystander cells irradiated at various stages in the cell cycle. Whereas a marked bystander effect for chromosomal aberrations occurred in V3 cells, the induction of chromosomal aberrations in irs3 bystander cells was minimal and similar to that of wild-type cells. Based on these findings, we hypothesize that HR is essential for the induction of SCE in bystander cells; however, HR is unable to repair the DNA damage induced in NHEJ-deficient bystander cells that leads to either SCE or chromosomal aberrations.