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 elucidating mechanisms of substrate choice by Pol ss. Elucidation of these mechanisms is critically and fundamentally important for understanding the molecular basis of mutation.

Public Health Relevance

The goal of this application is to understand the mechanisms of mutagenesis by DNA polymerases. This will result in fundamental knowledge regarding the basis of human diseases, as many of them are caused by mutations.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Radiation Therapeutics and Biology Study Section (RTB)
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Knowlton, John R
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Yale University
Schools of Medicine
New Haven
United States
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Sohl, Christal D; Ray, Sreerupa; Sweasy, Joann B (2015) Pools and Pols: Mechanism of a mutator phenotype. Proc Natl Acad Sci U S A 112:5864-5
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Eckenroth, Brian E; Fleming, Aaron M; Sweasy, Joann B et al. (2014) Crystal structure of DNA polymerase β with DNA containing the base lesion spiroiminodihydantoin in a templating position. Biochemistry 53:2075-7
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Eckenroth, Brian E; Towle-Weicksel, Jamie B; Sweasy, Joann B et al. (2013) The E295K cancer variant of human polymerase β favors the mismatch conformational pathway during nucleotide selection. J Biol Chem 288:34850-60
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Li, Yunlang; Gridley, Chelsea L; Jaeger, Joachim et al. (2012) Unfavorable electrostatic and steric interactions in DNA polymerase ýý E295K mutant interfere with the enzyme's pathway. J Am Chem Soc 134:9999-10010
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Berlow, Rebecca B; Swain, Monalisa; Dalal, Shibani et al. (2012) Substrate-dependent millisecond domain motions in DNA polymerase ýý. J Mol Biol 419:171-82

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