Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.

Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of ␥-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G 1 , greater impairment in S, and a substantial defect in late S/G 2 . Furthermore, the radiosensitivity of irs1SF cells is slight in G 1 but dramatically higher in late S/G 2 , while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G 2 , where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.DNA double-strand breaks (DSBs) are considered the most biologically damaging lesions produced by ionizing radiation (IR) and some chemicals. They also arise endogenously during DNA replication or as initiators of programmed processes, such as V(D)J recombination and meiotic exchange. If left unrepaired, DSBs can result in permanent cell cycle arrest, induction of apoptosis, or mitotic cell death caused by loss of genomic material (37); if repaired incorrectly, they can lead to carcinogenesis through translocations, inversions, or deletions (21, 67). Higher eukaryotic cells primarily repair DSBs by one of two genetically separable pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ repairs broken ends with little or no requirement for sequence homology and involves the XRCC4-LIG4 complex and the DNA-dependent protein kinase (DNA-PK) holoenzyme, consisting of the DNA end-binding heterodimer Ku70-Ku80 and the catalytic subunit DNA-PK cs (22,23,53). Cell lines defective in any of these genes are generally highly IR sensitive (Յ7-fold) and have marked deficiencies in DSB repair (9,28,40,69).HR, which appears to be less important than NHEJ for repairing IR-induced breaks in higher eukaryotes, utilizes extensive homology to faithfully restore the sequence at the break site by processes that involve proteins of the Rad52 epistasis group (20,61,62,63). In human cells, the main steps in HR are thought to be mediated by the single-strand binding protein RPA (3, 18, 52); the human homologs of Saccharomyces cerevisiae Rad51, Rad52, and Rad54 (4,56,66); and the Rad51 paralogs XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D (reviewed in r...

The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.Double-strand DNA breaks (DSBs) are produced by ionizing radiation (IR) and certain chemicals, and they likely occur frequently during DNA replication (21, 34). A single unrepaired DSB may stimulate cell cycle checkpoints and cause cell death (3, 25). Homologous recombination (HR) has emerged as a major DSB repair pathway in mammalian cells (29,35,44,65,66), as well as in the yeast Saccharomyces cerevisiae. Indeed, the analysis of radiosensitive yeast mutants has revealed a number of key genes involved in HR, which comprise the RAD52 epistasis group (2,32,54), and the HR pathway is conserved from yeast to humans (4,18,53,65). Although yeast is capable of proliferating at a reduced rate in the absence of functional HR, this repair pathway is essential for viability in cycling vertebrate cells for coping with DNA lesions arising during DNA replication (55,56,67,73). This species difference is probably due to the several-hundred-fold difference in genome size between vertebrates and yeast.ScRad51 is closely related to the Escherichia coli recombination protein RecA (5). Among the proteins of the Rad52 epistasis group, Rad51 has the highest degree of structural and functional conservation among all eukaryotes. The high degree of identity of ScRad51 with the human homolog (59% identity) and chicken homolog (59% identity) suggests that Rad51's function is conserved across eukaryotes. A central role for Rad51 in HR in vertebrates is supported by the finding that Rad51 deficiency (36, 55, 67), but not Rad52 or Rad54 deficiency, is lethal to cells (4,18,49,72). In vitro studies show that RecA and Rad51 form multimeric helical nucleoprotein filaments that are assembled on single-stranded DNA (ssDNA) (2). Recent work suggests that the preferred DNA substrat...

The phenotypically similar hamster mutants irs1 and irs1SF exhibit high spontaneous chromosome instability and broad-spectrum mutagen sensitivity, including extreme sensitivity to DNA cross-linking agents. The human XRCC2 and XRCC3 genes, which functionally complement irs1 and irs1SF, respectively, were previously mapped in somatic cell hybrids. Characterization of these genes and sequence alignments reveal that XRCC2 and XRCC3 are members of an emerging family of Rad51-related proteins that likely participate in homologous recombination to maintain chromosome stability and repair DNA damage. XRCC3 is shown to interact directly with HsRad51, and like Rad55 and Rad57 in yeast, may cooperate with HsRad51 during recombinational repair. Analysis of the XRCC2 mutation in irs1 implies that XRCC2's function is not essential for viability in cultured hamster cells.

The Chinese hamster ovary (CHO) cell mutant EM9 is hypersensitive to ethyl methanesulfonate (EMS) (10-fold) and ionizing radiation (1.8-fold), and it is unable to grow in medium containing chlorodeoxyuridine (CldUrd) under conditions in which 20% of genomic Thy is replaced by chlorouracil during DNA replication (9, 29). EM9 repairs -y-ray and EMS-induced single-strand breaks at a reduced rate and exhibits a 10-fold increase in the occurrence of sister chromatid exchange (SCE) (27,28). The sensitivity of EM9 to alkylating agents is suggestive of a defect in the base excision repair pathway, which involves sequential action by DNA glycosylase, apurinic-apyrimidinic endonuclease, deoxyribose-phosphodiesterase, DNA polymerase, and DNA ligase activities (17). The reduced rate of single-strand break rejoining suggests that the defect in EM9 lies within a postincision step of this pathway. EM9 is phenotypically similar to cells derived from individuals with Bloom's syndrome (BS), a cancer-prone autosomal recessive disorder characterized by high SCEs (10-fold) and sensitivity to alkylating agents (4,14,15 (31). As yet, no specific role has been identified for DNA ligase III (31). Altered DNA ligase activity in BS cells has been observed in several studies, in which it was proposed that the activity of DNA ligase I was affected (5, 6, 33, 34), but no major abnormality in DNA ligase activity was found in the one reported study in which EM9 was examined (7). However,