DNA polymerase ? (pol ?) is a DNA repair enzyme necessary for repairing damaged DNA that goes through the base excision repair (BER) pathway. In humans, pol ? repairs 20,000 DNA lesions per cell per day while only making a mistake once every 10,000 reactions. It has been found that approximately 30% of human tumors express mutants of pol ? that are not found in healthy tissue from the same individual. Some of these cancer-associated mutants still maintain the ability to repair DNA, but do so with less accuracy than the wild type. Structurally, these cancer-associated mutants are similar to the wild type allowing them to continue catalysis; however, the ability of the enzyme to differentiate between the right dNTP is greatly reduced. Crystallographic evidence suggests that pol ? follows an induced fit model and has a conformational change in the transferase domain upon binding of the correct nucleotide. It is proposed here that the conformational motions of the enzyme are necessary in the regulation of the fidelity of the enzyme, and that these conformational motions can be perturbed by mutations in the enzyme. The cancer-associated mutants also become sensitive to the upstream sequence of the template DNA, suggesting that the upstream sequence has an effect on the conformational changes in pol ? as well. In order to determine the effects on the confirmation motions of the enzyme, we will be using nuclear magnetic resonance (NMR) spectroscopy. NMR allows for atomic resolution of the enzyme in solution, and has been used extensively to study protein dynamics and structure. Using relaxation dispersion NMR experiments, R1? and Carr-Purcel-Meiboom-Gill (CPMG), the millisecond motions within pol ? can be determined, allowing for the measurement of changes in these motions caused by mutations in the enzyme and with changing template DNA substrates. The proposed project will help lead to a better understanding of the cancer-associated pol beta mutants role in tumorgenesis and tumor progression. This will help establish the role that pol plays in the formation and progress of human tumors, allowing for further research into finding new drug targets specific to mutant pol ?.
DNA polymerase beta (pol beta) is a human polymerase that is essential for repairing damaged DNA, thereby preventing cancer or cell death. Approximately 30% of human tumors have mutations in pol beta that either reduce the fidelity of the enzyme or completely knock out enzyme function. Here, I propose to measure how millisecond motions are related to the fidelity of pol beta in order to better understand how mutations in pol beta can lea to cancers in humans.
| 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 |
| Lisi, George P; East, Kyle W; Batista, Victor S et al. (2017) Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity. Proc Natl Acad Sci U S A 114:E3414-E3423 |
