Mechanism of completing cellular DNA replication

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The mechanism by which cells complete chromosome replication is not well understood. Yet the process must be remarkably efficient, occurring thousands of times per cell division in human cells, and is essential to maintain all cellular life and genome stability. This project will identify the processive molecular intermediates that occur during the completion of replication in cells. Students trained as part of this project will be qualified for careers in medicine, biotechnology, and research, and will include graduates and undergraduates at Portland State University, an urban college with a student body made up of more than 40% first generation college students. The project will also enhance early science and math education in the Portland public school system through a range of activities that include science fairs, science nights, career days, mentoring high school student research, and school-initiated science programs.In the bacterium Escherichia coli, completing replication involves an enzymatic system that limits cellular replication to its doubling point by allowing converging replication forks to transiently bypass each other before the excess, over-replicated regions are incised, resected, and joined. The reaction initiates through the combined action of SbcCD-ExoI and requires RecBCD to join convergent strands of the replication fork. In mutants where completing replication is impaired, cell viability becomes dependent on an aberrant form of recombination that also drives genetic instabilities. While these observations clearly establish the importance of this fundamental process, the substrates and mechanism by which these enzymes catalyze the reaction remain unknown. This research uses a bi-directionally replicating plasmid mini-chromosome to identify the structural intermediates that occur in wild type cells and mutants impaired for the completion reaction, in vivo. The project will determine how chi and ter sequences, which are known to regulate completion enzymes, affect the reaction in vivo using genomic profiling and plasmid mini-chromosomes. The research additionally employs a genetic screen designed to identify and characterize additional enzymes involved in the completion process.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.