We have recently shown that DNA polymerase beta (Pol ss) plays a key role in maintaining genomic stability and preventing cellular transformation. Approximately 30% of human tumors studied express Pol ss variants. We have shown that several of these tumor-associated variants of Pol ss induce cellular transformation in immortalized cells by a mutational mechanism. Pol ss functions in base excision repair (BER) to fill in gaps that are created during the excision of DNA damage that arises at the rate of 20,000 lesions per cell per day. Thus, accurate DNA synthesis by Pol ss is critical for the maintenance of genomic stability and the prevention of human cancer. The broad, long-term objective of the proposed research is to characterize the fidelity mechanisms of DNA polymerase beta at a molecular level.
The specific aims are to test the hypothesis that amino acid residues of Pol ss that are critical for accurate DNA synthesis are located within regions of Pol ss other than the hydrophobic hinge, loop II, and the region between HhH motifs and to study mechanisms used by specific regions of Pol ss that are critical for accurate DNA synthesis. We will take a combined genetic, biochemical and biophysical approach to address these hypotheses. We will use transient-state kinetics to characterize Pol beta mutator mutants that we have obtained via genetic screens. We will also employ fluorescence assays to characterize how the motion of the Pol beta protein affects fidelity. Finally, crystal structures of various Pol beta mutator mutants will be solved in order to relate our biochemical findings to enzyme structure.

Public Health Relevance

The goal of this application is to determine why enzymes that copy DNA make mistakes. The mistakes made by these enzymes can become mutations and lead to cancer. Learning more about how these enzymes copy DNA and make mistakes will help us design new strategies to prevent and treat human cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA080830-13
Application #
8018179
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Okano, Paul
Project Start
1999-04-07
Project End
2014-02-28
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
13
Fiscal Year
2011
Total Cost
$315,935
Indirect Cost
Name
Yale University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Eckenroth, Brian E; Towle-Weicksel, Jamie B; Nemec, Antonia A et al. (2017) Remote Mutations Induce Functional Changes in Active Site Residues of Human DNA Polymerase ?. Biochemistry 56:2363-2371
Alnajjar, Khadijeh S; Garcia-Barboza, Beatriz; Negahbani, Amirsoheil et al. (2017) A Change in the Rate-Determining Step of Polymerization by the K289M DNA Polymerase ? Cancer-Associated Variant. Biochemistry 56:2096-2105
Alnajjar, Khadijeh S; Negahbani, Amirsoheil; Nakhjiri, Maryam et al. (2017) DNA Polymerase ? Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the ?-? Bridging Group of the Incoming dNTP. Biochemistry 56:5449-5456
Mahmoud, Mariam M; Schechter, Allison; Alnajjar, Khadijeh S et al. (2017) Defective Nucleotide Release by DNA Polymerase ? Mutator Variant E288K Is the Basis of Its Low Fidelity. Biochemistry 56:5550-5559
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
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
Ray, Sreerupa; Menezes, Miriam Rose; Senejani, Alireza et al. (2013) Cellular roles of DNA polymerase beta. Yale J Biol Med 86:463-9
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
Eckenroth, Brian E; Towle-Weicksel, Jamie B; Sweasy, Joann B et al. (2013) The E295K cancer variant of human polymerase ? favors the mismatch conformational pathway during nucleotide selection. J Biol Chem 288:34850-60

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