The degradation of proteins is known to be an essential regulatory mechanism vital for biological processes and diseases including cell cycle progression, DNA repair, and cancer. While the majority of cellular proteins are stable, there are a small percentage of cellular proteins that are unstable, temporal acting, and function in a regulatory manner. An example of these are the Escherichia coli proteins UmuD' (UmuD) and UmuC. UmuD'C comprise an error-prone DNA polymerase, DNA polV, necessary for the survival of the bacterium following extensive DNA damage. The Umu proteins are part of a DNA damage tolerance system termed SOS mutagenesis. Recent work suggests that the mammalian counterparts of these prokaryotic error-prone polymerases may play a role in processes from cancer establishment to induction of somatic cell hypermutation of immunoglobulin genes. Therefore, DNA polV activity and regulation is an ideal model to study for two reasons: (i) the regulation of DNA polV is vital to the understanding of the SOS mutagenic response and will provide a scientific foundation for exploring the regulation of eukaryotic error-prone polymerases, and (ii) DNA polV is a highly suitable model for studying selective protein degradation, a process known to be vital for cell integrity. The mechanisms by which proteases selectively recognize these and other unstable proteins are unknown and determining these mechanisms is important in understanding how SOS mutagenesis and other biological processes are specifically regulated. It is also unknown how starvation conditions, such as those experienced by bacterial cells in stationary phase and in their natural environment, affects the protease-substrate interaction. The research proposed in this project will elaborate on the mechanism(s) of Umu instability and will address the processing of UmuD (and potentially the other Umu proteins) demonstrated during stationary phase growth. We will use numerous molecular, biochemical, and genetic techniques to achieve the following aims: 1. To test DNA polV function and regulation during stress conditions not known to induce significant DNA damage. 2. To evaluate the complexity of the degradation signals of the Umu proteins and their effect on regulation. ? ?
