The Roles DNA Polymerase Epsilon in DNA Replication, Repair and Cell Cycle Checkpoints

The Scripps Research Institute, La Jolla CA

Investigators

Abstract

Maintenance of genomic integrity is of utmost importance to living organisms. An intricate system of enzymes has evolved to deal with the tremendous task of replicating chromosomes once and only once per cell division cycle while maintaining genetic information with great accuracy. Among those enzymes are a group of DNA polymerases which catalyze the polymerization of nucleotides during the replication process. However, those enzymes are associated with a large complex of accessory protein known collectively as " the replication complex". The components of that complex are required not only for DNA replication, but also for editing errors, organizing topology, and regulating progression through the cell division cycle. The mechanisms that restrict cell cycle progression, including mitosis, when DNA is incompletely or improperly replicated are known as checkpoints. DNA polymerase epsilon (Pol epsilon), one of the three major replicative polymerases, appears to play roles in all of these processes. Defects in those processes can lead to an increased mutation rate and genomic instability. This project is directed at understanding the roles of Pol epsilon. This enzyme is involved in DNA replication, repair, checkpoint regulation of cell cycle progression and in cell viability. It is not unexpected that, as a major replicative polymerase, Pol epsilon is essential. Yet, surprisingly, recent evidence has shown that the essential function of this enzyme does not require its enzymatic activity but instead involves regions of the protein of unknown function. This project will extend those studies to elucidate the essential function of this domain of the protein and, thereby, the essential function of DNA Pol epsilon. The experiments employ a wide array of molecular, genetic, and cell biological techniques including recombinant DNA technology, protein biochemistry, mass spectrometry, flow cytometry, chromatin immunoprecipitation, and targeted proteolysis, among others. The project will employ both postdoctoral trainees and graduate students, training them in eukaryotic microbial genetics, biochemistry and modern molecular and cell biology. It is anticipated that the discoveries made in the context of this research will contribute to the general population in the form of improved understanding of a basic biological process, DNA replication.