Studies on Mechanisms of DNA Rearrangements in Mammalian Cells.

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(1) Molecular analysis of genes for homologous recombination and cell growth from fission yeast Schizosaccharomices pombe. We have isolated S. pombe genes for homologous to budding yeast S. cereviseae DST2 gene, whose product have the activity of DNA strand exchange. S. pombe genomic library was screened with a probe from S. cereviceae DST2 and nucleotide sequences of positive clones were determined. Approximately 3 kb open leading frame which encodes 991 amino acids was found in the resulted clones and is called dhp1 (DST2 homologue of S. p-ombe). The product had the activity of DNA strand exchange and was essential for the mitotic cell growth. Furthermore we also isolated another homologue of the DST2, which is called dhp2. In contrast to the dhpl, the dhp2 is not essential for mitotic cell growth. The DST2 homologues will be used to analyze the mechanism of homologous recombination in S. pombe.(2) Cloning and analysis of gene (s) for homologous recombination in mammalian cells. The … More result of amino acid sequence of the dhpl gene was used to isolate human CDNA for homologous recombination and we succeeded it. We are analyzing the structure and function of this human CDNA and its product. We are also trying to isolate mouse genes for homologous recombination and to construct mutant mice which are defective in the gene for homologous recombination.(3) In vivo and vitro study of homologous recombination of SV40-derived shuttle vector DNAs in mammalian cells. SV40-derived shuttle vectors which contain direct repeats in the both sides of the galK^+ marker were incubated with an extract from HeLa cytosol in the presence of SV40 large T antigen. The vector DNAs were assayed for galK deletion formation using E. coligal^-. Using this assay system, the formation of galk deletion was detected at a high frequency and is dependent on the presence of both SV40 origin of DNA replication and SV40 large T antigen. This in vitro system will become a powerful tool for elucidating biochemical aspects of homologous recombination in mammalian cells.(4) Role of DNA topoisomerases on illeg, inmate recombination. We found that calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoiscimerase, which we have previously indicated. Furthermore, we have also analyzed the effects of topoisomerase inhibitors in mammalian cultured cells. The DNA topoisomerase II inhibitor VM26 stimulated deletion formation in SV40-derived DNA in mon]Cey cells, implicating that topoisomerase II participates in deletion formation in mammalian cells. 10. KEY WORDS Less