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.

Public Health Relevance

Cancer is ultimately caused by genomic mutations, which in turn can result from damaged DNA. The goal of this project is to determine why mutated forms of an enzyme which is responsible for repairing DNA can themselves cause cancer by comparing the way the normal enzyme and the mutated enzymes behave at various stages of the DNA repair process. These studies will ultimately illuminate the vital steps of the normal enzyme, which is one part of one of the first cellular lines of defense against cancer.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F09-P (20))
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Jakowlew, Sonia B
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Yale University
Schools of Arts and Sciences
New Haven
United States
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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