MECHANISMS OF HOMOLOGOUS RECOMBINATION IN HUMAN CELLS

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The multi-step process of malignant transformation not only involves mutations in dominant genes, but also the elimination of the function of suppressor genes. Mitotic recombination between homologous genes is one mechanism by which mammalian cells become homozygous for such recessive genes. However, the mechanisms involved in such recombination and the factors that can affect its frequency are not yet known in detail. Since mitotic recombination is a rare event, we utilize a model system involving a substrate for detecting intrachromosomal homologous recombination between two homologous genes stably integrated into the genome of human cells. We prepared two sets of matched strains that carry the recombination substrate. The sets differ in the target genes, and each is composed of cell strains proficient or completely deficient in nucleotide excision repair or O6-methyl-guanine (O6-MeG) repair. With them we showed that carcinogens induce homologous recombination and that it is stimulated by unexcised DNA damage, rather than by excision repair. We propose to use these strains, and additional strains to be constructed, to study other cellular factors affecting the frequency of recombination. The questions we will address include: 1) At what phase in the cell cycle human cells are most sensitive, and does this depend on the type of agent or repair capacity? 2) What is the relationship between DNA adducts located in the region of the recombination substrate, interference with DNA replication, and the induction of recombination? 3) Does the level of transcription of the recombination substrate genes affect the frequency? 4) Is O6-MeG responsible for inducing recombination? And if so, how? 5) Can inhibition of human cell topoisomerase I stimulate homologous recombination. And if so, does this involve long-lived double- strand breaks or single-strand breaks? 6) Does carcinogen-induced homologous recombination introduce errors (mutations), and if so, could these arise as a result of mismatched bases in a heteroduplex? 7) Will Bloom's syndrome cells exhibit an abnormally elevated frequency of intrachromosomal homologous recombination comparable to their elevated frequencies of sister chromatid exchanges and chromosome abnormalities?