Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low. We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis. This error-avoiding process is catalysed by a protein encoded by the mutT gene of Escherichia coli, mutations of which increase the occurrence of A.T----C.G transversions 100 to 10,000 times the level of the wild type. Spontaneous oxidation of dGTP forms 8-oxo-7,8-dihydro-2'-dGTP (8-oxodGTP), which is inserted opposite dA and dC residues of template DNA with almost equal efficiency, and the MutT protein specifically degrades 8-oxodGTP to the monophosphate. This indicates that elimination from the nucleotide pool of the oxidized form of guanine nucleotide is important for the high fidelity of DNA synthesis.

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 ...

Hypothalamic peptide hormones regulate the secretion of most of the anterior pituitary hormones, that is, growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and adrenocorticotropin. These peptides do not regulate the secretion of prolactin, at least in a specific manner, however. The peptides act through specific receptors, which are referred to as seven-transmembrane-domain receptors or G-protein-coupled receptors. Although prolactin is important in pregnancy and lactation in mammals, and is involved in the development of the mammary glands and the promotion of milk synthesis, a specific prolactin-releasing hormone has remained unknown. Here we identify a potent candidate for such a hormone. We first proposed that there may still be unknown peptide hormone factors that control pituitary function through seven-transmembrane-domain receptors. We isolated the complementary DNA encoding an 'orphan' receptor (that is, one for which the ligand is unknown). This receptor, hGR3, is specifically expressed in the human pituitary. We then searched for the hGR3 ligand in the hypothalamus and identified a new peptide, which shares no sequence similarity with known peptides and proteins, as an endogenous ligand. We show that this ligand is a potent prolactin-releasing factor for rat anterior pituitary cells; we have therefore named this peptide prolactin-releasing peptide.

No abstract

Oxidized guanine (8-oxo-7,8-dihydroguanine; 8-oxo-G) is a potent mutagen because of its ambiguous pairing with cytosine and adenine. The Escherichia coli MutT protein specifically hydrolyzes both 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-rGTP), which are otherwise incorporated in DNA and RNA opposite template A. In vivo, this cleaning of the nucleotide pools decreases both DNA replication and transcription errors. The effect of mutT mutation on transcription fidelity was shown to depend on oxidative metabolism. Such control of transcriptional fidelity by the ubiquitous MutT function has implications for evolution of RNA-based life, phenotypic expression, adaptive mutagenesis, and functional maintenance of nondividing cells.

No abstract

No abstract

8-0xguanie nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies.Once 8-oxodGTP is formed in the cellular nucleotide po-, this mutagenic nucleotide is incorporated into DNA and would cause transversion mutations. The MutT protein ofEscherichia coli possesses enzyme activity to hydrolyze 8-oxodGTP to the corresponding nucleoside monophosphate and thus may be responsible for preventing the occurrence of such mutations. Here we show that the human cell has an enzyme specifically hydrolyzing 8-oxodGTP in a fashion similar to that seen with MutT protein. The human 8-oxodGTPase has been found in cell-free extracts from Jurkat cells and purified >400-fold.Analyses by gel filtration and gel electrophoresis revealed that the molecular mass of the native form of human 8-oxodGTPase is 18 kDa. Mg2+ ion is required for the enzyme action and the optimum pH for the reaction is pH 8.0. The enzyme hydrolyzes 8-oxodGTP to 8-oxodGMP with a K.m value of 12.5 pM. dGTP and dATP are also degraded to dGMP and dAMP, respectively, with Km values 70 times greater than that for 8-oxodGTP. dTTP and dCTP are not hydrolyzed. These properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.Mutator mutants that show an increased frequency of spontaneous mutations have led to elucidation of the multiple pathways of spontaneous mutagenesis. Studies on Escherichia coli mutator genes and their products revealed that a major cause of spontaneous mutation is errors of DNA replication and that the cell possesses multistep mechanisms to correct such errors (1). In addition, certain types of spontaneous DNA damage would cause mutations (2) and the cell comes equipped with mechanisms to repair such damage.Among 15 known E. coli mutator genes, 12 are shown to be involved in correction of replicational errors and/or spontaneous DNA damage (1, 3-6). Recently we obtained evidence that the mutT mutator gene is involved in a hitherto unknown mechanism for reducing spontaneous mutation frequency (7).A mutT mutator mutant shows a frequency of A-T -COG transversion 100-10,000 times the level of the wild type (8).The MutT protein specifically prevented misincorporation of dGMP onto poly(dA)/oligo(dT)20 template DNA in vitro (9).This antimutagenic effect of MutT protein appeared to be catalytic and was achieved through its action on dGTP but not on DNA or DNA polymerase (H.M. and M.S., unpublished results). We and others noted that the MutT protein has a weak nucleoside triphosphatase activity with a substrate preference to dGTP (9, 10). Subsequently we found that the nucleotide that is misincorporated opposite the dA residue of the template is not dGMP but rather its oxidized form, 8-oxodGMP. When 8-oxodGTP was added to an in vitro DNA replication system, 8-oxodGMP was incorporated opposite dA and dC residues of the template, with almost e...