DNA REPLICATION, REPAIR, AND MUTAGENESIS IN EUKARYOTIC AND PROKARYOTIC CELLS

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Studies on the mechanism of prokaryotic mutagenesis have focused on the roles of the RecA and UmuD, C-like mutagenesis proteins (which facilitate bypass of lesions by DNA polymerase during replication of damaged DNA). Biochemical assays have revealed that UmuD, its cleavage product (UmuD'), and the functionally homologous, plasmid-encoded MucA' proteins physically interact with RecA. This interaction may provide a mechanism by which the Umu-like proteins are targeted to lesions in DNA. Two E. coli strains have been constructed that allow the identification of Umu phenotypes based upon a simple phenotypic complementation assay. These strains have facilitated the cloning of three new umu-like operons from R-plasmids R391, R446b and R471a. These tester strains were also utilized to identify several novel plasmid- encoded umuC mutants. In studies on mammalian DNA repair, we determined the subcellular distribution and regulation by UV light of a 127 kDa protein component of a primate UV-damaged DNA-binding (UV- DDB) complex. Structural homologs of this UV-DDB protein were identified in slime mold and rice, and a partial cDNA of a Drosophila homolog was isolated. New evidence supports an important role for the UV-DDB complex in mammalian DNA damage-recognition and repair: One additional Xeroderma Pigmentosum patient ("XP variant") with a defect in UV-DDB activity was identified (4 patients are now known to lack this damage-recognition activity); the recovery of UV-DDB activity is delayed in UV-irradiated cells from XP groups A, D, and C patients, which correlates well with their DNA repair-deficiency; and cells from mammalian tissues that do not normally express UV-DDB activity are more UV- sensitive than their clonal isolates which have regained the binding activity. In studies on SV40 as a model eukaryotic replicon, we focused on the viral small t-antigen. In a purified in vitro SV40 DNA replication system, we found that small-t inhibits large T antigen- dependent replication. However, in vivo, small-t enhances SV40 DNA replication. Infection experiments also indicate that small-t stimulates progression of permissive monkey cells - but not non-permissive rodent cells - from the G0/G1 to the S phase of the cell cycle, presumably leading to an optimal intracellular environment for viral replication. In rodent experiments, small-t mutants preferentially transformed rapidly proliferating lymphoid cells and appeared unable to transform cells that normally have a low mitotic rate, such as mesothelial cells. In these experiments, we also found that wild-type SV40 induces mesotheliomas in hamsters. Subsequently, we have found that more than 60% of human mesotheliomas contain and express SV40-like sequences.