The long term goal of this research is to understand the role of the umuDC gene products of Escherichia coli in the molecular mechanism of induced mutagenesis and in the control of the cell cycle in response to DNA damage. UmuD and UmuC have been shown to permit DNA polymerase III to carry out translesion synthesis which results in mutations (44). Recent results from the Walker laboratory have shown that intact UmuD (the form inactive in translesion synthesis) together with UmuC participate in a cell cycle checkpoint mechanism in response to DNA damage (Opperman, T., S. Murli, and G. C. Walker, manuscript in preparation). Preliminary results from the Walker laboratory suggest that UmuD and UmuD interact physically with the epsilon and beta subunits of DNA polymerase III. Experiments outlined in this proposal are designed to provide insights into how the interaction of UmuD, UmuD and UmuC with these two subunits of DNA polymerase III, and with other cellular proteins is related to their roles in SOS mutagenesis and cell cycle checkpoint control. I will biochemically characterize protein- protein interactions using a variety of techniques including gel filtration and affinity chromatography, a co-immunoprecipitation, chemical cross-linking, and surface plasmon resonance (BIAcore). Subsequent experiments will concentrate on obtaining biochemical evidence of the UmuD and UmuC checkpoint control in vitro.
