It is generally accepted that recombinational repair is an important aspect of DNA damage processing in prokaryotes and yeast. However, few studies have directly assessed whether shared pathways for DNA repair and genetic recombination exist in mammalian cells. Recently, the cloning of mammalian genes complementing DNA repair-deficient phenotypes and the discovery of their potential relationships to RAD genes in Saccharomyces cerevisiae has revealed the prospect of addressing the issue of shared roles for DNA repair genes in both DNA repair and mitotic recombination pathways in mammalian cells. This issue is of particular interest and importance to carcinogenesis since loss of heterozygosity, a hallmark of malignant conversion in many cancers, can occur through mitotic recombination mechanisms. In this application, they intend to investigate the role of a mammalian DNA repair/recombination gene, ERCC1, in mitotic recombination and repair in mammalian cells. The nature of a number of different mutations in the ERCC1 gene will be determined by DNA sequencing after reverse transcription polymerase chain reaction amplification of RNA from CHO cell ERCC1 mutants. CHO cell lines in which the ERCC1 gene disrupted or otherwise altered by targeted homologous recombination will be generated and characterized with respect to repair and recombination phenotypes. These """"""""knock-out"""""""" mutants will be engineered by gene transfer methods to contain heteroallelic gene duplications designed for the testing of specific hypotheses regarding whether the ERCC1 gene is involved in mitotic recombination and targeted recombination in mammalian cells, and how the mechanisms of spontaneous and induced mitotic recombination in mammalian cells may resemble those of yeast. An ERCC1 minigene plasmid will be used to complement knock-out mutants, and site-directed and random mutagenesis techniques will be used to specifically alter regions of ERCC1 prior to complementation. Epistatis between ERCC1 and other repair genes will be investigated. These experimental approaches will determine what role the ERCC1 repair gene may play in pathways of mitotic recombination in mammalian cells, and will increase our understanding of DNA repair and recombination in mechanisms of genome alteration and carcinogenesis.
