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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumComplex formation by the human RAD51C and XRCC3 recombination repair proteinsJean-Yves Masson*, Alicja Z.Stasiak†, Andrzej Stasiak†, Fiona E.Benson‡, and Stephen C.West*§ *Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom; †Laboratoire d’Analyze Structural, Université de Lausanne, 1015 Lausanne, Switzerland; and ‡Lancaster University, Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster LA1 4YQ, United Kingdom In vertebrates, the RAD51 protein is required for genetic recombination, DNA repair, and cellular proliferation. Five paralogs of RAD51, known as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, have been identified and also shown to be required for recombination and genome stability. At the present time, however, very little is known about their biochemical properties or precise biological functions. As a first step toward understanding the roles of the RAD51 paralogs in recombination, the human RAD51C and XRCC3 proteins were overexpressed and purified from baculovirus-infected insect cells. The two proteins copurify as a complex, a property that reflects their endogenous association observed in HeLa cells. Purified RAD51C-XRCC3 complex binds single-stranded, but not duplex DNA, to form protein-DNA networks that have been visualized by electron microscopy. Homologous recombination plays an important role in the repair of DNA double-strand breaks (DSBs) caused by ionizing radiation or from the breakdown of stalled replication forks. Accurate DSB repair, using the sister chromatid as a template, is necessary for the maintenance of genome stability, and defects in this process can lead to the introduction of mutations, chromosomal translocations, apoptosis, and cancer. The RAD51 protein promotes recombination by catalyzing the invasion of the broken ends of the DSB into the intact sister chromatid. RAD51 is a structural and functional homolog of Escherichia coli RecA and forms helical nucleoprotein filaments in which the DNA lies extended and underwound. Filaments form preferentially on tailed duplex DNA substrates that mimic the resected DSBs thought to be present at chromosomal break sites (1, 2). Strand invasion by RAD51 is stimulated by RAD52, RAD54, and RP-A, resulting in the formation of a heteroduplex joint (2–11). Yeast that are defective in RAD51 exhibit reduced levels of recombination and are sensitive to ionizing radiation, but the cells remain viable. In contrast, disruption of RAD51 in the mouse is lethal (12, 13). Moreover, inactivation of a RAD51 transgene in chicken cells leads to chromosome fragmentation followed by cell death (14). These observations emphasize the essential role that RAD51 and recombinational repair play in normal cellular proliferation. Such an extreme phenotype, however, has not been observed after disruption of RAD52 (15, 16) or RAD54 (17, 18). Although recombination proteins such as RAD51, RAD52, and RAD54 have been well conserved from yeast to vertebrates, it is not clear whether there are vertebrate homologs of yeast Rad55 and Rad57. Defects in RAD55 and RAD57 result in radiation sensitivity, a phenotype that can be partially complemented by overexpression of Rad51 (19). Biochemical studies of Rad55 and Rad57 have shown that the two proteins form a heterodimer that interacts with Rad51 and stimulates Rad51-mediated pairing reactions (20). It is therefore thought that Rad55/57 play an accessory role in strand invasion, possibly displacing RP-A during nucleoprotein filament assembly by RAD51. The repair-defective phenotype of rad55/57 mutants is elevated at reduced temperatures (21), a property that is often associated with proteins that are composed of multiple subunits or are participants in large multiprotein complexes (22). Rad55 and Rad57 are known to share limited amino acid similarity with Rad51 and may have been derived by duplication of the ancestral gene encoding Rad51. However, apart from their conserved ATP binding motifs and the ability of Rad55/57 to catalyze ATP hydrolysis (20), they are clearly divergent in function from Rad51. Direct homologs of the yeast RAD55 and RAD57 genes have not been identified in vertebrates. However, five genes that bear a distant resemblance to RAD51 have been identified, and, like Rad55 and Rad57, their products have been classified as members of the RAD51 family (23). The first members of this class, encoded by the XRCC2 and XRCC3 genes (24, 25), were identified by genetic complementation of the repair-deficient irs1 and irs1SF rodent cell lines (26, 27). Furthermore, three distinct genes designated RAD51B (also known as RAD51L1, hREC2, or R51H2), RAD51C (a.k.a. RAD51L2), and RAD51D (a.k.a. RAD51L3 or R51H3) were identified by database analyses on the basis of their sequence homology to RAD51 (28–32). All five RAD51 paralogs share limited (≈20– 30%) amino acid sequence identity with RAD51, much of which is concentrated around the two Walker ATP binding sites (23, 33). Multiple protein alignments of the RAD51 family members suggest that RAD51D and XRCC3 are closest to yeast Rad57, whereas XRCC2 is more homologous to yeast Rad55 (23, 34). The RAD51 family members are required for normal levels of recombination and DSB repair. Whereas cells defective in XRCC2 (irs1) and XRCC3 (irs1SF) are moderately sensitive to x-rays or γ-radiation (≈2 fold), they display an extreme sensitivity (60- to 100-fold) to DNA cross-linking agents such as cisplatin, nitrogen mustard, or mitomycin C (25, 35). The mutant cell lines also exhibit a high incidence of spontaneous and mutagen-induced chromosomal aberrations (36) and show defects in chromosome segregation (37). Moreover, both irs1 and irs1SF show a significant (100- and 25-fold, respectively) decrease in the frequency of DSB repair by homologous recombination (38, 39). In recent studies, the XRCC2 gene was targeted in the mouse, and disruptions were found to confer an embryonic lethal phenotype (40). XRCC2−/− blastocysts showed a genetic instability phenotype, with high levels of chromosomal aberrations and a sensitivity to γ-rays. They also exhibited developmental defects in the nervous system, indicating a potential role for This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: ssDNA, single-stranded DNA; DSB, double-strand break; mAb, monoclonal antibody; pAb, polyclonal antibody.
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