Rad51 proteins share both structural and functional homologies with the bacterial recombinase RecA. The human Rad51 (HsRad51) is able to catalyse strand exchange between homologous DNA molecules in vitro . However the biological functions of Rad51 in mammals are largely unknown. In order to address this question, we have cloned hamster Rad51 cDNA and overexpressed the corresponding protein in CHO cells. We found that 2-3-fold overexpression of the protein stimulated the homologous recombination between integrated genes by 20-fold indicating that Rad51 is a functional and key enzyme of an intrachromosomal recombination pathway. Cells overexpressing Rad51 were resistant to ionizing radiation when irradiated in late S/G2phase of the cell cycle. This suggests that Rad51 participate in the repair of double-strand breaks most likely by homologous recombination involving sister chromatids formed after the S phase.

DNA polymerase ␤ (pol ␤) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol ␤ cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol ␤. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol ␤ overexpression in cancer predisposition and tumor progression during chemotherapy.

Rad51 protein plays an essential role in recombination repair of DNA double-strand breaks and DNA crosslinking adducts. It is part of complexes which can vary with the stage of the cell cycle and the nature of the DNA lesions. During a search for Rad51-associated proteins in CHO nuclear extracts of S-phase cells by mass spectrometry of proteins immunoprecipitated with Rad51 antibodies, we identified a centrosomal protein, c-tubulin. This association was confirmed by the reverse immunoprecipitation with c-tubulin antibodies. Both proteins copurified from HeLa cells nuclear extracts following a tandem affinity purification of double-tagged Rad51. Immunofluorescence analysis showed colocalization of both Rad51 and c-tubulin in discrete foci in mammalian cell nuclei. The number of colocalized foci and their overlapping area increased in the presence of DNA damage produced by genotoxic treatments either during S phase or in exponentially growing cells. These variations did not result from an overall stress because microtubule cytoskeleton poisons devoid of direct interactions with DNA, such as taxol or colcemid, did not lead to an increase of this association. The recruitment of Rad51 and c-tubulin in the same nuclear complex suggests a link between DNA recombination repair and the centrosome function during the cell cycle.

DNA replication is an asymmetric process involving concurrent DNA synthesis on leading and lagging strands. Leading strand synthesis proceeds concomitantly with fork opening, whereas synthesis of the lagging strand essentially takes place on a single-stranded template. The effect of this duality on DNA damage processing by the cellular replication machinery was tested using eukaryotic cell extracts and model DNA substrates containing site-specific DNA adducts formed by the anticancer drug cisplatin or by the carcinogen N-2-acetylaminof luorene. Bypass of both lesions was observed only with fork-like substrates, whereas complete inhibition of DNA synthesis occurred on damaged singlestranded DNA substrates. These results suggest a role for additional accessory factors that permit DNA polymerases to bypass lesions when present in fork-like DNA.The opposing orientation of the two DNA strands in the double helix imposes an asymmetry upon the replication process (1). Since polymerases only synthesize DNA in one direction (5Ј 3 3Ј), one strand (the leading strand) is synthesized continuously in the direction in which the helix unzips, whereas the other (the lagging strand) is synthesized discontinuously in the opposite direction in numerous short pieces known as Okazaki fragments. Therefore, leading strand synthesis proceeds concomitantly with fork opening from a double-stranded template, whereas synthesis of the lagging strand essentially takes place on a single-stranded template. Single-stranded and fork-like DNA templates have been previously suggested as models for lagging and leading strand replication, respectively (2, 3), although current models of DNA replication propose a mechanism in which leading and lagging strand replication are coupled. Genotoxic agents that induce bulky lesions in DNA often hamper progression of the replication fork and result in either nonor miscoding nucleotides. In a process known as translesion synthesis (TLS), the replication machinery will eventually proceed through some lesions with an increased error rate, resulting in the induction of mutations (4 -6). We wished to investigate the effect of the asymmetry of DNA replication on the efficiency of translesion synthesis. For this purpose, we have compared TLS in vitro by eukaryotic cell extracts on forked or single-stranded (ss) DNA templates containing single site-specific adducts formed by a chemotherapeutic agent (cisplatin) or a strong chemical carcinogen (N-2-acetylaminof luorene or AAF). For both adducts, we observed TLS only when the adduct was located in the forked template. MATERIALS AND METHODSMaterials. The DNA damage-inducible CDK inhibitor p21 protein, purified as described (7), was a generous gift from Prof. Bernard Ducommun (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France). Calf thymus DNA polymerases ␣, ␦, and were purified as reported (8). All oligonucleotides were synthesized on a Cyclone Plus DNA synthesizer from MilliGen͞Biosearch and purified on denaturing 20% polyacrylamide gels. [...

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Bacteriophage Mu dX(Ap lac) was used to isolate a mutation in an Escherichia coli lexA(Def) strain representing a previously undescribed gene (dinY) which does not seem to be under the direct control of LexA. The insertion created a dinY::lacZ fusion in which beta-galactosidase expression required a DNA-damaging treatment (UV irradiation or mitomycin) and activable RecA protein. This strain showed a decreased Weigle reactivation of bacteriophage lambda. However, it was fully inducible for UV mutagenesis. Two-dimensional gel electrophoresis analysis identified two spots absent in the mutant which were both UV inducible only in the presence of activated RecA protein (RecA*). This finding suggests that the dinY::lacZ fusion lies in a gene either that is under the direct control of activated RecA or whose product undergoes RecA*-dependent posttranscriptional/posttranslational modification(s). The dinY gene may also control the expression of some other gene(s) and/or lie in an operon. The fusion was mapped at a position between 41 and 41.5 min on the E. coli chromosome, in the vicinity of the ruv operon.