The E. coli. umu operon encodes two mutagenic proteins (UmuD,C) which facilitate bypass of lesions by DNA polymerase during replication of damaged DNA. Functional homologues of UmuD,C, e.g., MucA,B, are found on naturally occurring plasmids. We found that the post-translational processing of UmuD protein to its mutagenically active form, UmuD', is relatively inefficient. MucA, which is highly mutagenic, is processed very efficiently. UmuD and UmuD'proteins physically interact with RecA protein, and this interaction may provide a mechanism by which the Umu proteins are targeted to DNA lesions. Construction of a delta(umuDC) mutation showed that the Umu D,C proteins, normally required for inducible mutagenesis, are not essential for cell survival. Bacterial strains carrying the deletion have been constructed which permit the rapid cloning of umuDC-like genes. We have also studied the recognition and removal of mammalian DNA lesions. In primate cells, we identified, characterized, and determined the primary sequence of, a novel nuclear UV damage-specific DNA binding (UV-DDB) protein. This protein, of relatively recent evolutionary origin, is inducible to high levels by UV. The UV-DDB protein may be involved in early steps of DNA repair (e.g., lesion recognition) based on its high affinity for UV damaged DNA in vitro, its regulation by UV light in vivo, and its absence in some patients with the heritable UV repair deficiency, xeroderma pigmentosum (group E). We have cloned and sequenced the gene encoding the UV-DDB protein; a structural defect in this gene may be responsible for XP-E. In studies on DNA replication, we utilized the DNA virus, SV40. The role of the SV40 oncogene, large T antigen (Tag) is well known in viral and cellular DNA replication. However, a second SV40 gene product, small t antigen (t), is enigmatic. This protein is required for Tag-mediated induction of cellular DNA replication and transformation when nondividing cells are infected by SV40, but t is not required in proliferating cells. We have identified a biochemical function for t: It inhibits Tag-mediated viral DNA replication in vitro. We also found that tdelta mutants transform rapidly proliferating cell types (e.g., lymphocytes), but not cells with a low proliferative rate (e.g., fibroblasts). Small t may, by inhibiting the cellular phosphatase, PP2A, alter the phosphorylation state of Tag required for its roles both in viral and cellular DNA replication. Tag is also known to bind and inactivate several antioncogene products, e.g., Rb, p53, and p3OO. This interaction may also be affected by t.
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