Dynamic Characterization of Cancer-Associated Variants of DNA Polymerase Beta

Yale University, New Haven CT

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Abstract

DESCRIPTION (provided by applicant): Cancer is ultimately caused by damage to the genome. DNA is constantly subjected to chemical and environmental assaults, resulting in approximately 20,000 damaged base pairs per cell per day. Left unrepaired, these lesioned sites could result in mismatched-induced mutations upon replication, eventually causing cancer. Fortunately, cells possess highly effective processes which detect, remove, and replace such lesions. One of the crucial enzymes in this process is DNA Polymerase beta (Pol beta), which is responsible for the fidelity-ensuring step of inserting the appropriate nucleotide into a gap generated by prior excision of a damaged base pair. Accordingly, Pol beta mutants with reduced fidelity or activity relative to normal or wild-type (WT) Pol beta cause cancer. Although many such mutants have been biochemically characterized, the ways in which minor structural changes to WT Pol beta can dramatically alter Pol beta's function still remain unclear. During the process of DNA repair, Pol beta undergoes a series of large-scale, reversible conformational changes. These changes have traditionally been assumed to occur only in the presence of substrate. Recently, however, we and others have found that, along all stages of the catalytic pathway, WT Pol beta undergoes rapid, reversible conformational changes on its own, even in the absence of substrate. Surprisingly, these dynamic motions appear to sample conformations formed at later stages of the catalytic pathway. We hypothesize that the dynamic behaviors observed in WT Pol beta are altered in cancer-associated variants. This goal of this project is to test this hypothesis by characterizing the dynamics of mutagenic Pol beta variants using solution-state relaxation dispersion NMR techniques and to compare the results of these studies with both the dynamics of WT Pol beta and with previous studies which have characterized the sorts of mutations each variant induces. Mutant forms of Pol beta have been found in over 30% of all human cancer cells. Many of these mutants have reduced fidelity or activity relative to WT Pol beta. This study will help explain how point mutations in Pol beta can result in mutagenic variants.