MUTAGENESIS AND REPAIR OF DNA

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The long term goals of this project are to understand the molecular basis of UV and chemical mutagenesis, to understand the roles of the E. coli UmuD and UmuC proteins in this process, and to use the knowledge base accumulated for this system to study issues in protein-structure function relationships, and protein-protein interactions. The molecular mechanism of UV and chemical mutagenesis is not yet known in any organism yet is central to an understanding of the basis of chemical carcinogenesis and of the origin of some human genetic diseases. This proposal takes advantage of the power and relative ease of E. coli genetics, molecular biology, and biochemistry to address this central unsolved problem in nucleic acid biology. Biochemical methods will be used to study the influence of UmuC, UmuD, and other proteins on the behavior of DNA polymerases on DNA templates carrying a defined chemical lesion at a known position. We will also attempt to determine functions of the UmuD and UmuC proteins by looking for activities suggested by their similarity to bacteriophage T4 DNA polymerase accessory proteins and by using an assay based on DNA substrates carrying a large inverted repeat. In collaboration with Greg Petsko's and Dagmar Ringe's laboratories, an effort will be made to determine the crystal structure of UmuD+. Monocysteine derivatives of UmuD and UmuD+ will be constructed and used to study the conformation and interactions of these proteins. Genetic and biochemical techniques will be used to analyze the interactions of UmuD and UmuC with other proteins. A set of mutant UmuD and UmuD + proteins will be analyzed biochemically to determine the nature of their defects with respect to homodimer formation, interaction with RecA, autodigestion capability, and interaction with UmuC. Certain physiological phenomenon we have identified have the potential to offer insights into UmuD and UmuC and will be analyzed further; these include the inhibition of SOS-induction caused by an increased dosage of umuDC and umuDC-dependent inhibition of DNA replication. We will test genetically whether umuDC function is essential for cell viability.