The mouse Rad5l gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad5l null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad5Sl/-) pups among 148 neonates examined but a few Rad5-/-embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad5l null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.Genetic recombination leads to new associations of genetic elements. In meiosis, recombination between closely paired homologous chromosomes results in extensive reshuffling of paternal and maternal genes, and the progeny can be better fitted to cope with the environment. Recombination occurring in somatic cells is manifested as sister chromatid exchange and the outcome, by itself, does not alter the cellular genotype.Molecular mechanisms of recombination have been studied extensively in bacteria and lower eukaryotes. The recA gene of Escherichia coli plays an essential role in recombination as well as in DNA repair and induction of SOS functions (1-3). The RecA protein has the potential to promote homologous pairing and strand exchange of DNA in the presence of adenosine 5'-triphosphate (ATP) (2-6). In yeast Saccharomyces cerevisiae, RAD51, RAD52, and RAD54 genes, belonging to the RAD52 epistasis group, were initially identified as those involved in the repair of DNA damage induced by ionizing radiation (7,8), and subsequently were shown to be responsible for mitotic recombination (9-12). Among them the RAD51 gene is a homologue of the E. coli recA gene and plays crucial roles in both mitotic and meiotic recombination as well as in repair of double-strand breaks of Isolation of Targeted ES Cell Clones. The ES cell line CCE was cultured on a feeder cell layer and electroporated, using 5 X 107 cells and 50 ,tg of the linearized targeting vector DNA, as described (25,26). Colonies doubly resistant to G418 (250 ,ug/ml) and ganciclovir (5 ,uM) were selected and expanded on feeder layers in 24-well plates. Homologous recombinants were identified by Southern blot analysis of restriction enzymedigested DNA. DNAs were prepared from cells cultured in the absence of feeder cells and subjected to Southern blot analysis.The DNA (8 jig) was cleaved with BamHI, subjected to agarose gel electrophoresis, blotted onto Hybond N+ membrane (Amersham), and hybridized to probe A. To ensure targeted disruption of the Rad5l gene, the DNA was digested with EcoRV or HindIll, followed by hybridization with ...

The possibility that Escherichia coli MutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OHdATP), oxidized forms of dATP, but not (R)-8,5-cyclodATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OHdGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OHdGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2. However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.Endogenous oxidation of DNA and DNA precursors by reactive oxygen species appears to induce spontaneous mutations, aging, and various diseases, including cancer and neurodegeneration (1, 2). 8-OH-dGTP 1 is an oxidized form of dGTP and induces A:T to C:G transversions because it can pair with adenine as well as cytosine (3-6). It is known that the Escherichia coli MutT protein hydrolyzes 8-OH-dGTP to 8-hydroxydGMP (4). Because the mutation rate in a mutT-deficient strain increases up to 1000-fold as compared with the wild type (7), 8-OH-dGTP is considered to be a major source of spontaneous mutations caused by endogenous reactive oxygen species, and MutT appears to efficiently prevent the spontaneous occurrence of A:T to C:G transversion mutations. In human cells, the hMTH1 protein is considered to be a functional homologue of the E. coli MutT because the hMTH1 protein hydrolyzes 8-OH-dGTP in vitro and suppresses the mutator phenotype of E. coli mutT-deficient cells (8, 9).Recently, we found that 2-hydroxy-dAdo and 2-OH-dATP are produced efficiently by reactive oxygen species treatment of dAdo and dATP, respectively (10, 11). 2-OH-dATP specifically induces G:C to T:A transversion mutations and is more mutagenic than 8-OH-dGTP in vivo (5). Thus, 2-OH-dATP is thought to act as an endogenous mutagen in cells. However, the presence of a hydrolyzing activity for 2-OH-dATP has not been described. We supposed that the MutT and hMTH1 proteins may act on this mutagenic nucleotide, 2-OH-dATP. We report here that the hMTH1 protein, which is known as an 8-OHdGTPase, hydrolyzes 2-OH-dATP more efficiently than 8-OHdGTP. In addition, hMTH1 also hydrolyzed 8-OH-dATP, another oxidized form of dATP, as efficiently as 8-OH-dGTP. On the other han...

The three-dimensional structure of Escherichia coli 3-methyladenine DNA glycosylase II, which removes numerous alkylated bases from DNA, was solved at 2.3 A resolution. The enzyme consists of three domains: one alpha + beta fold domain with a similarity to one-half of the eukaryotic TATA box-binding protein, and two all alpha-helical domains similar to those of Escherichia coli endonuclease III with combined N-glycosylase/abasic lyase activity. Mutagenesis and model-building studies suggest that the active site is located in a cleft between the two helical domains and that the enzyme flips the target base out of the DNA duplex into the active-site cleft. The structure of the active site implies broad substrate specificity and simple N-glycosylase activity.

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