The long range objective of our studies is to understand the mechanism by which carcinogens cause malignant transformation of human calls. There is now strong evidence. drawn from several different fields, that carcinogenesis is a multistepped process. One or more steps may involve somatic cell mutations. However, recent genetic evidence, obtained from molecular biology studies of cells derived from persons with hereditary retinoblastoma, indicates that mitotic recombination between allelic genes can lead to the development of tumors by making the cell homozygous for the mutated allele, which permits expression of a recessive trait. Little is known about the mechanism of genetic recombination in mammalian cells. We recently demonstrated that chemical and physical carcinogens can induce homonomous recombination between chromosomal genes in mammalian cells (Wang et al., Mol. Cell. Biol, 8, 196-202, 1988). The question we intend to address in the proposed research are: How do these carcinogens induce recombination? What mechanisms are involved? Is recombination stimulated by the presence of carcinogen-induced damage interfering with DNA replication during S-phase? Alternatively, is recombination stimulated by the introduction of breaks in DNA caused by excision repair processes, or by single stranded regions of parental DNA generated during repair? What role do DNA ligases play in recombination? Are cells from cancer prone individuals more sensitive than cells from normal persons? We propose to integrate into the chromosome of human cells a single copy of a plasmid containing two copies of a gene coding for a selectable genetic marker. Each gene is interrupted at a unique site by an 8 bp linker site for a restriction endonuclease so that a productive recombinational event is required to get a functional (selectable) gene product. The plasmids will be stably integrated into the genome of human cells that differ in their DNA repair capacity, including cells derived from patients with xeroderma pigmentosum ataxia telangiectasia, and Bloom's syndrome, as well as cells from normal persons.
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