DNA Polymerase Beta and Mutagenesis

University Of Arizona, Tucson AZ

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Abstract

PROJECT SUMMARY (NO CHANGE FROM FUNDED APPLICATION) Mutations result in human disease and arise from both endogenous and exogenous damage to DNA. Mutations can also result from misincorporation errors produced by DNA polymerases. The focus of the proposed project is on the structure and function of DNA polymerase beta (Pol ß), a key enzyme in base excision repair (BER). The major role of BER is the repair of bases in DNA that are damaged by reactive oxygen and nitrogen species (RONS) arising from predominantly endogenous sources. It is estimated that greater than 20,000 DNA adducts per cell per day are repaired by BER and it is known that aberrant BER results in mutations and genomic instability. Previous work and research from our laboratory during the current funding period shows that mutator variants of Pol ß, which catalyze error-prone DNA synthesis, predominantly harbor alterations of specific amino acid residues distant from its active site. This suggests that accurate DNA synthesis by Pol ß is governed by amino acid residues distant from its active site. Pol ß is comprised of four domains called 8 kD, thumb, palm, and fingers. The active site for the dRp lyase activity of Pol ß is in the 8 kD domain, whereas the thumb, palm, and fingers act to bind DNA, contain the polymerase active site, and bind to dNTP, respectively. Importantly, the mutator variants we have identified and characterized map to all domains of Pol ß. These results imply that Pol ß employs a number of molecular mechanisms to ensure correct dNTP substrate choice by Pol ß. Presteady-state biochemical characterization of these mutator variants shows that they are deficient in discrimination of the correct from the incorrect dNTP predominantly at ground state binding of the dNTP substrate, although one of the mutators is deficient in discrimination during nucleotidyl transfer. How residues at distances from the active site and in different domains of Pol ß influence substrate choice is a focus of this application. The broad, long-term objective of the proposed research is to characterize the catalytic mechanism of Pol ß. The first specific aim is to characterize the catalytic mechanism of correct dNTP incorporation by Pol ß. The second specific aim is to test the hypothesis that the kinetic pathway of Pol ß for incorporation of incorrect dNTPs differs from that for correct dNTPs. A combined genetic, biochemical, and biophysical approach will be used to accomplish these aims with the goal of eludating mechanisms of substrate choice by Pol ß. Elucidation of these mechanisms is critically and fundamentally important for understanding the molecular basis of mutation.