The long-term goal of this research is to understand how organisms accurately duplicate their genetic material by coordinating the actions of their DNA replication machinery with those of other cellular proteins involved in repair, damage tolerance, and cell cycle progression. Failure of an organism to do so can have catastrophic consequences ranging from disease, such as cancer, to death. The proposed research program utilizes both biochemical and genetic approaches to understand the roles played by the beta processivity clamp of the E. coli replicative DNA polymerase in coordinating DNA replication, repair, damage tolerance, and cell cycle progression. The beta clamp participates in a DNA damage checkpoint control as well as in translesion DNA synthesis (TLS). In addition, it interacts with a variety of proteins known to function in various aspects of DNA metabolism. As one Aim, we will test our hypothesis that unique interactions of beta with the different umuDC gene products influence the choice between replication, checkpoint, and TLS. These studies will have broad relevance to understanding integration of replication and TLS in both prokaryotic and eukaryotic cells. As a second Aim, we will test our proposal that beta participates in at least one DNA repair and/or umuDC-independent damage tolerance function by determining the molecular basis of the UV light sensitivity of a dnaN59 beta mutant. These studies will further our understanding of mechanisms that couple replication and repair. As a third Aim, we will test our hypothesis that certain partner proteins bind to overlapping surfaces on beta, and that by competing with each other for binding to the clamp, beta is able to regulate which proteins gain access to the replication fork. These studies will provide important insights into fundamental mechanisms of polymerase switching and replication fork management. In summation, we anticipate that the study of the coordinated regulation of DNA replication, DNA damage tolerance and repair, and cell cycle progression in E. coli will provide a valuable framework for understanding these same processes in humans, where the complexity of the events is far greater.
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