Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ).Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54 -/-, KU70 -/-and RAD54 -/-/KU70 -/-, generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing γ-radiation-induced DSBs during G 1 -early S phase while recombinational repair is preferentially used in late S-G 2 phase. RAD54 -/-/KU70 -/-cells were profoundly more sensitive to γ-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54 -/-and RAD54 -/-/KU70 -/-cells, with RAD54 -/-/KU70 -/-cells exhibiting significantly higher levels of chromosomal aberrations than RAD54 -/-cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.

Yeast rad51 mutants are viable, but extremely sensitive to γ-rays due to defective repair of double-strand breaks. In contrast, disruption of the murine RAD51 homologue is lethal, indicating an essential role of Rad51 in vertebrate cells. We generated clones of the chicken B lymphocyte line DT40 carrying a human RAD51 transgene under the control of a repressible promoter and subsequently disrupted the endogenous RAD51 loci. Upon inhibition of the RAD51 transgene, Rad51 -cells accumulated in the G 2 /M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase-arrested Rad51 -cells carried isochromatid-type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.

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...

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Sister chromatid exchange (SCE) frequency is a commonly used index of chromosomal stability in response to environmental or genetic mutagens. However, the mechanism generating cytologically detectable SCEs and, therefore, their prognostic value for chromosomal stability in mitotic cells remain unclear. We examined the role of the highly conserved homologous recombination (HR) pathway in SCE by measuring SCE levels in HR-defective vertebrate cells. Spontaneous and mitomycin C-induced SCE levels were significantly reduced for chicken DT40 B cells lacking the key HR genes RAD51 and RAD54 but not for nonhomologous DNA end-joining (NHEJ)-defective KU70 ؊/؊ cells. As measured by targeted integration efficiency, reconstitution of HR activity by expression of a human RAD51 transgene restored SCE levels to normal, confirming that HR is the mechanism responsible for SCE. Our findings show that HR uses the nascent sister chromatid to repair potentially lethal DNA lesions accompanying replication, which might explain the lethality or tumorigenic potential associated with defects in HR or HR-associated proteins.Symmetrical exchanges between newly replicated chromatids and their sisters can be visualized cytologically in vertebrate cells if the DNA of one chromatid is labelled with 5-bromodeoxyuridine (BUdR) during synthesis. Sister chromatid exchanges (SCEs) can be induced by various genotoxic treatments (10), suggesting that SCEs reflect a DNA repair process. Cytological assessment of SCE levels in peripheral blood lymphocytes is used as an index of the mutagenic potential of environmental factors. More importantly, ϳ10 SCEs occur spontaneously in normally cycling human cells (5, 8), suggesting a link between SCE and DNA replication. Elevated spontaneous SCE levels are observed in cells from Bloom syndrome patients (9), in mouse cells that lack poly(ADP-ribose) polymerase (29) or KU70 (15), and in hamster cells with defects in XRCC1 (28), but the causal relationships between these enzymes and SCE are not clear.While the phenomenon of SCE has long been established (27) and many observations about the induction of SCEs have been made, their molecular basis remains obscure. SCE is intimately associated with DNA replication, and eukaryotic cells exposed to DNA-damaging agents in G 2 show elevated SCE levels only after completing a subsequent replication cycle (32). Homologous recombination (HR) was suggested as one of the mechanisms responsible (13,14). While HR occurs between sister chromatids in yeast as a means to replicate around UV-induced lesions (12), it has not been considered constitutively active during metazoan mitosis, perhaps because of the predominance of the nonhomologous DNA end-joining (NHEJ) pathway (30). In addition, the lack of recombinational repair mutants precluded direct testing of HR's involvement in SCE, so other models evolved. It was proposed that SCEs result from strand switching at stalled replication forks (20). Another model involved topoisomerase II action at coincident breaks at replication for...

Mice carrying transgenic rearranged V region genes in their IgH and Igkappa loci to encode an autoreactive specificity direct the emerging autoreactive progenitors into a pre-B cell compartment, in which their receptors are edited by secondary Vkappa-Jkappa rearrangements and RS recombination. Editing is an efficient process, because the mutant mice generate normal numbers of B cells. In a similar nonautoreactive transgenic strain, neither a pre-B cell compartment nor receptor editing was seen. Thus, the pre-B cell compartment may have evolved to edit the receptors of autoreactive cells and later been generally exploited for efficient antibody diversification through the invention of the pre-B cell receptor, mimicking an autoreactive antibody to direct the bulk of the progenitors into that compartment.

REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.Both prokaryotic and eukaryotic cells are endowed with multiple specialized DNA polymerases that are devoid of 3Ј35Ј proofreading exonuclease activity and replicate undamaged DNA in vitro with low fidelity and weak processivity (5). These specialized enzymes support DNA synthesis past a spectrum of template strand base damage by a process called translesion DNA synthesis (TLS), a mode of DNA damage tolerance that is fundamental to the survival of cells that suffer arrested DNA replication associated with damage to DNA.REV1 protein (which is confined to the eukaryotic kingdom) is a member of the Y family of DNA polymerases (14, 21). However, in vitro, the nucleotidyl transferase activity of REV1 is limited to the incorporation of just one or two dCMP moieties in a template-directed manner, regardless of the template nucleotide composition (19,30). This catalytic activity supports TLS past sites of base loss in vitro (19) and conceivably subserves this function in vivo. However, REV1 protein is also required for mutagenesis in both yeast and mammalian cells exposed to DNA-damaging agents that are not associated with the generation of sites of base loss, such as UV radiation (14). Remarkably, the dCMP transferase activity is dispensable for this function (1,14,18). Indeed, inactivation of the dCMP transferase activity in yeast does not result in defects in DNA damage-associated mutagenesis (9). Furthermore, a yeast mutant strain with a missense mutation in the N-terminal BRCT domain of REV1 retains dCMP transferase activity in vitro, even though it is deficient in TLS past sites of base loss and photoproducts (18).Several laboratories have demonstrated that the C-terminal ϳ100 amino acids of both mouse REV1 (mREV1) and human REV1 proteins can interact with multiple specialized DNA polymerases implicated in TLS (6,17,20,27). Additionally, different specialized DNA polymerases can compete with one another for binding to REV1 in vitro (6). Collectively, these observations suggest a presently unknown role(s) for REV1 in TLS that is unrelated to its dCMP transferase function.REV1 protein colocalizes with proliferating cell nuclear antigen (PCNA) in replication factories (27) and binds to other members of the Y family of DNA polymerases, to which it belongs, in...

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.