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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM099990-01
Application #
8221655
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
2012-01-15
Project End
2015-12-31
Budget Start
2012-01-15
Budget End
2012-12-31
Support Year
1
Fiscal Year
2012
Total Cost
$276,437
Indirect Cost
$98,687
Name
Yale University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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