Polymerase Dynamics at the Replication Fork

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Intellectual Merit. DNA replication is a central process important for proliferation of all cell types. Because of highly conserved features, the bacterium Escherichia coli provides a useful prototype to understanding replication of all organisms. DNA replication proceeds bidirectionally on anti-parallel duplex DNA at a branch point called the replication fork. Because of this, one strand is synthesized as short fragments (Okazaki fragments) on one strand (the lagging strand) at the fork. Since the replicase that synthesizes DNA is highly processive, meaning that it can proceed long distances without releasing, there must be a mechanism for triggering the lagging strand replicase to release and recycle to the RNA primer for the next Okazaki fragment. This research seeks to distinguish between two models for Okazaki fragment cycling. The favored hypothesis is that synthesis of a new RNA primer provides a cycling signal sensed by the DnaX complex that loads both a ring-like processivity factor on DNA and chaperones the polymerase component of the replicase onto it. The DnaX complex is also thought to chaperone the polymerase off of the old Okazaki fragment and escort it onto the new primer, concomitant with loading a new processivity factor and hydrolysis of ATP. Macromolecular interactions responsible for this chaperoning reaction will be determined. The results of the research will make a significant contribution to our understanding of the dynamics of DNA replication and the role of this important process in the maintenance of genome stability, not just in bacteria, but also in higher organisms.Broader Impacts. The project will involve graduate and undergraduate students in the research. A laboratory module, incorporating hands-on use of robotics equipment for screening for replication mutants, will be implemented for an undergraduate biochemistry course, and details of the module will be published, so that other universities will be able to use it for their curricula. Such training will provide valuable exposure to equipment and procedures commonly used in pharmaceutical and biotechnology industries, but rarely made available in undergraduate classes, and thus may increase the competitiveness of the students to obtain employment upon graduation. The PI will disseminate fundamental knowledge on bacterial DNA replication to the scientific and lay public through continued contributions to an updatable online encyclopedia article published in collaboration with the American Society for Biochemistry and Molecular Biology.