Regulation of DNA Replication and Repair

The long-term goal of this research program is to develop an integrated mechanistic view of how organisms coordinate the actions of their replication machinery with those of other cellular factors involved in DNA repair and damage tolerance. Failure to do so leads to a loss of genetic fidelity and contributes to human disease. Furthermore, inappropriate regulation of Y-family DNA polymerase function is proposed to contribute to mutations that lead to cancer. Work from our laboratory and others have demonstrated unambiguously that DNA polymerase processivity clamps (¿ or DnaN sliding clamps) play critically important roles in this complex process. The proposed research program utilizes an integrated genetic¿biochemical¿physical biochemical approach, placing particular emphasis on determining how the ¿ clamp helps to coordinate the actions of the E. coli replicase, DNA polymerase III holoenzyme (Pol III), with the dinB-encoded Pol IV, which acts in translesion DNA synthesis (TLS), and with the Hda protein, which helps to regulate initiation of DNA replication by inactivating the DnaA initiator protein. We will utilize in vitro assays to characterize interactions of Pol III and Pol IV with various mutant ¿ clamp proteins. As part of this work, we will purify heterodimeric clamp proteins bearing either a single mutation in one subunit, or different mutations in each subunit. Using these mutant clamps, we will dissect the mechanism by which the ¿ clamp mediates a switch between Pol III and Pol IV to coordinate high fidelity replication with TLS. We will complement these studies with genetic analyses. We anticipate that the model(s) for polymerase switching supported by our results will serve as a valuable paradigm for similar switch mechanisms in other organisms, including humans. In addition, since Y-family Pols are remarkably well conserved throughout all three branches of life, results from our studies will also contribute significantly to our understanding of mechanisms underlying mutagenesis under times of stress, thereby impacting on pathogenesis and antibiotic resistance, as well as mechanisms contributing to immunoglobulin diversity by error-prone replication during somatic hypermutation. We will also apply the approaches that we have developed to characterize polymerase switching to Hda protein in order to understand the role of the ¿ clamp in coordinating replication with Hda-dependent regulation of initiation of replication. Failure to properly regulate initiation leads to over-replication, genome instability, and can be lethal. We will distinguish between different models for Hda function, and will determine whether Hda and Pol III simultaneously bind to the same ¿ clamp. We will also determine whether Hda acts to limit access of Y-family Pols to the replication fork until such time as they are required for TLS. Finally, since replication errors contribute significantly to mutagenesis, and since the coordinate regulation of initiation and elongation of DNA replication is critically important for genome stability, our findings in these areas may also identify new classes of targets for the development of novel antibiotics.