I " ; Analyses of the Roles of the UmuDC Proteins of E . coli in SOS Mutagenesis and Cell Cycle Regulation

Regulated mechanisms that inhibit DNA synthesis and cell cycle progression in response to DNA damage have been shown to be essential for DNA damage tolerance in eukaryotes. Analogous mechanisms have not been as well defined in prokaryotes. Evidence presented in this thesis suggests that the UmuD and UmuC proteins of Escherichia coli participate in a mechanism to inhibit growth and DNA synthesis upon exposure to DNA damaging treatments, thereby increasing cell survival. This hypothesis grew out of the study of umuDC-mediated growth inhibition at 30°C (cold sensitivity). The umuDC operon is a member of the SOS regulon, a group of twenty or more genes whose expression is coordinately induced by DNA damaging treatments. The UmuD' and UmuC proteins have been well characterized for their activity in SOS mutagenesis, the mutagenesis resulting from exposure to DNA damaging agents such as UV irradiation. Constitutive expression of the umuDC operon from a multicopy plasmid confers cold sensitivity for growth which is associated with an inhibition of DNA synthesis. The genetic requirements for observing umuDC-mediated cold sensitivity are distinct from those for SOS mutagenesis supporting that these are distinct functions of the umuDC gene products. High levels of intact UmuD, the form inactive in SOS mutagenesis, confer growth inhibition at 30°C in combination with UmuC. Further analyses revealed that the umuDC gene products, expressed at physiologically relevant levels, inhibit the transition from stationary phase to exponential growth of UV irradiated stationary phase cells and increase UV resistance, possibly by allowing time for DNA repair processes to be completed. This is most likely the result of the inhibition by the umuDC gene products of a Fis-dependent activity. Suppression of the UV sensitivity of a AumuDC strain by afis mutation demonstrates that the inhibition of afis-dependent activity is central to the UV resistance conferred by the umuDC gene products. Intact UmuD and UmuC increase cell survival after UV irradiation. This previously uncharacterized activity of UmuD and UmuC is correlated with the inhibition of DNA synthesis after UV irradiation. The umuC125 mutation, which specifically interferes with the ability of UmuC to confer growth inhibition at 30'C without interfering with SOS mutagenesis, also abolishes the ability of UmuC to regulate DNA synthesis after UV irradiation. This suggests that this novel activity of intact UmuD and UmuC is functionally related to umuDC-mediated growth inhibition at 30°C. Thesis supervisor: Graham C. Walker Title: Professor of Biology