Decoupling of DNA unwinding and synthesis in the replisome induces genome instability

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The long-held textbook viewpoint on DNA replication is that the replisome is a stable multiprotein complex assembly that seamlessly coordinates coupled synthesis on the leading and lagging strands. However, recent evidence indicates a more dynamic replisome, where individual protein components exchange during replication, creating stochastic processes of DNA unwinding, priming, and synthesis. A more dynamic replisome would be useful in responding to various roadblocks or challenges to DNA replication progression. Still, enzymatic coordination at the replication fork must be maintained, otherwise one activity may dominate, creating excess and dangerous replication intermediates. This project will determine the molecular contacts between replisome proteins that act to coordinate and control DNA unwinding with synthesis. Undergraduate and graduate students will be trained in techniques of advanced enzyme kinetics, precise genetic manipulations, and novel biochemical and cellular assays. Scientific outreach programs will encourage local elementary school students to explore the wonders of their own genome through "DNA Days". By definition, the advancement of the replisome is dependent on the DNA duplex unwinding activity of the hexameric helicase to create single-strand templates. Once it is unwound, the polymerase holoenzyme rapidly resynthesizes duplex DNA. This project will integrate complex in vitro biochemistry experiments with in vivo genomic editing and correlated cellular assays to characterize and quantify multicomponent molecular contacts required to maintain replisome coupling. This research will also determine and differentiate coupled kinetic enzymatic processes from those that are linked directly through multisubunit replisome contacts. The resulting data, methods, and findings will be broadly distributed and will provide deeper insights into the mechanisms for replisome control and coordination of multiple enzymatic activities necessary for proper genome maintenance.This research is funded by the Genetic Mechanisms program in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.