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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA080830-16
Application #
8696423
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Knowlton, John R
Project Start
1999-04-07
Project End
2019-02-28
Budget Start
2014-05-05
Budget End
2015-02-28
Support Year
16
Fiscal Year
2014
Total Cost
$310,728
Indirect Cost
$94,844
Name
Yale University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Towle-Weicksel, Jamie B; Dalal, Shibani; Sohl, Christal D et al. (2014) Fluorescence resonance energy transfer studies of DNA polymerase ?: the critical role of fingers domain movements and a novel non-covalent step during nucleotide selection. J Biol Chem 289:16541-50
Eckenroth, Brian E; Fleming, Aaron M; Sweasy, Joann B et al. (2014) Crystal structure of DNA polymerase ? with DNA containing the base lesion spiroiminodihydantoin in a templating position. Biochemistry 53:2075-7
Gridley, Chelsea L; Rangarajan, Sneha; Firbank, Susan et al. (2013) Structural changes in the hydrophobic hinge region adversely affect the activity and fidelity of the I260Q mutator DNA polymerase *. Biochemistry 52:4422-32
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
Murphy, Drew L; Jaeger, Joachim; Sweasy, Joann B (2011) A triad interaction in the fingers subdomain of DNA polymerase beta controls polymerase activity. J Am Chem Soc 133:6279-87
Yamtich, Jen; Starcevic, Daniela; Lauper, Julia et al. (2010) Hinge residue I174 is critical for proper dNTP selection by DNA polymerase beta. Biochemistry 49:2326-34
Yamtich, Jennifer; Sweasy, Joann B (2010) DNA polymerase family X: function, structure, and cellular roles. Biochim Biophys Acta 1804:1136-50
Lin, George C; Jaeger, Joachim; Eckert, Kristin A et al. (2009) Loop II of DNA polymerase beta is important for discrimination during substrate binding. DNA Repair (Amst) 8:182-9
Yamtich, Jennifer; Speed, William C; Straka, Eva et al. (2009) Population-specific variation in haplotype composition and heterozygosity at the POLB locus. DNA Repair (Amst) 8:579-84
Dalal, Shibani; Starcevic, Daniela; Jaeger, Joachim et al. (2008) The I260Q variant of DNA polymerase beta extends mispaired primer termini due to its increased affinity for deoxynucleotide triphosphate substrates. Biochemistry 47:12118-25

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