The long-term goals of this project are to characterize and understand how conformational motions in DNA polymerase ? (Pol ?) participate in its catalytic reaction. Pol ? is an essential enzyme in the repair process of DNA, a process that maintains the integrity of the human genome and ultimately protects against cancer. To advance the current understanding of this enzyme, solution NMR experiments in combination with steady- state and pre-steady-state kinetics will be brought to bear on Pol ? with focus on three specific aims. 7 To determine if WT Pol ? motions in the apo, binary, and ternary complexes correlate with rate constants observed for the catalytic reaction sequence and to determine at what step these motions occur. 7 To test the hypothesis that tumor-associated Pol b mutations (I260M, K289M &E295K) have altered millisecond motions compared to the WT enzyme. 7 To characterize how WT and cancer-associated mutants, described in Aim 2, differentially interact with oxidatively damaged DNA.
Aim 1 will be met by solution NMR relaxation dispersion experiments on enzyme complexes that mimic discreet steps on the Pol ? catalytic reaction coordinate.
In Aim 2, Pol ? mutations that are known to be associated with human cancer will be investigated by enzyme kinetics, and NMR dynamics measurements to elucidate at what stage on the reaction coordinate altered motions or conformations occur. Lastly, Aim 3 will compare enzyme kinetics experiments that monitor the incorporation of dNTPs opposite oxidatively damaged DNA with NMR dynamics experiments. These experiments will focus on differences between WT and cancer- related mutations in their enzyme kinetics parameters and conformational changes when faced with damaged DNA.

Public Health Relevance

Human DNA is constantly being damaged this damage must be repaired to prevent mutations that would lead to cancer. DNA polymerase ? is an enzyme that fixes damaged DNA. This proposal aims to understand how polymerase b works, to provide insight that will lead to better drugs.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function E Study Section (MSFE)
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Barski, Oleg
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Yale University
Schools of Arts and Sciences
New Haven
United States
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Liptak, Cary; Mahmoud, Mariam M; Eckenroth, Brian E et al. (2018) I260Q DNA polymerase ? highlights precatalytic conformational rearrangements critical for fidelity. Nucleic Acids Res 46:10740-10756
Moscato, Beth; Swain, Monalisa; Loria, J Patrick (2016) Induced Fit in the Selection of Correct versus Incorrect Nucleotides by DNA Polymerase ?. Biochemistry 55:382-95
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