Structure-function Studies Of DNA Polymerases

Environmental Health Sciences

Investigators

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Abstract

Summary of Work: We are examining the relationship between the structures of DNA polymerases and their functions, including fidelity. Accomplishments this year include the following. We determined that the frameshift fidelity of human DNA polymerase lambda is very low. This is consistent with a proposed role for DNA polymerase lambda in repairing strand breaks using DNA ends with limited base pairing homology. This hypothesis was supported with studies of strand break repair in extracts of human cells and with our determination of the X ray crystal structure of human DNA polymerase lambda. We determined the efficiency and fidelity of bypass of abasic sites, 8-oxo-guanine and thymine dimers by Y family DNA polymerases Sso Dpo4 and human DNA polymerase eta and by the model bacteriophage T7 replicative DNA polymerase. These studies are revealing the strategies used by cells to replicate DNA damaged by environmental agents in a manner that avoids mutagenesis and cytotoxicity and the accompanying adverse health consequences. Background: Multiple DNA polymerases exist in organisms from bacteria to humans, in families designated A, B, C, X, Y or RT (for Reverse Transcriptase). We now know that human cells harbor at least 15 template-dependent DNA polymerases. The precise, and perhaps redundant, functions of most of these are unknown, providing a rich opportunity for study. This project investigates the relationship between the structures of DNA polymerases and their properties, with emphasis on fidelity. Much of our recent effort has focused on polymerases discovered since 1999, especially those in families Y and X. Our general approach is to purify recombinant enzymes and to describe the fidelity with which they synthesize DNA in vitro, and then try to understand the mechanisms responsible for avoiding and generating errors using structural information to guide our thinking and hypotheses.