Oxidative stress is caused by a number of environmental agents such as polycyclic aromatic hydrocarbons like dioxin, heavy metals, pesticides, and ultraviolet and ionizing radiation. Reactive oxygen species are created by normal cell processes and in elevated levels by oxidative stress. They cause multiple types of DNA lesions, the most common being 8-oxo-guanine (8-oxo-dG). This lesion is highly mutagenic due to its ability to base pair with the incorrect base adenine in a manner that escapes detection by the proofreading activity of the replicative polymerases. It is also blocks DNA synthesis by the replicative polymerases 1, 4 and 5, which have difficulty synthesizing past most DNA damage. Translesion synthesis (TLS) by specialized polymerases of the Y-family is therefore a necessary component of DNA replication. Direct bypass of damage allows completion of DNA replication in the face of lesions that would otherwise stall the replication fork, collapse of which can lead to strand breaks and other gross chromosomal changes. TLS past several DNA lesions by Y-family members occurs with higher efficiency compared to replicative polymerases, making them the preferred choice for ensuring replication is completed, even though the fidelity of many TLS polymerases is relatively low. Bypass of the UV-light induced thymine-thymine dimer is performed by the Y-family member polymerase 7 and occurs with high efficiency but somewhat low fidelity, with errors generated during 3-5% of bypass events. These errors can be detected and removed by replicative polymerase proofreading activities, allowing pol 7 multiple chances to insert the correct base, if needed. This allows the overall pol 7 dependent bypass reaction to suppress mutations caused by UV light. A similar logic has been proposed for the suppression of mutagenesis during pol 7 dependent bypass of 8-oxo-dG. Recently, we demonstrated that human pol 7 does indeed perform highly efficient bypass of 8-oxo-dG, but also observed that the bypass occurred with remarkably low fidelity. Errors were generated during ~50% of all bypass events. The most frequent error was adenine misinsertion opposite 8-oxo-dG, which as noted above frequently goes undetected by the proofreading activities of replicative polymerases due to its structural similarity to a correct base pair. This implies that the mechanism by which pol 7 suppresses mutagenesis caused during 8-oxo-dG bypass differs from that of UV-light induced damage. We hypothesize that the observed suppression of mutagenesis by pol 7 during 8-oxo-dG bypass occurs by an increase in the fidelity of the polymerase by association with additional replication proteins.
We aim to determine which factors may influence the fidelity of the bypass reaction and also to determine what role, if any, pol 7 plays in the generation of mutations during times of oxidative stress when the number of 8-oxo-dG bypass events would be greatly elevated. The goals of this proposal are: 1) to determine the effects of replication proteins on the efficiency and fidelity of 8-oxo-dG bypass by human pol 7;2) to reconstitute and characterize the 'complete'8-oxo-dG lesion bypass reaction in vitro;3) to identify and characterize human pol 7 mutants, including known SNPs, that display altered fidelity for 8-oxo-dG bypass;and 4) to determine the mutation rate of wild type and pol 7 deficient cells, and cells expressing mutant forms of pol 7, under conditions of oxidative stress. The long term objectives of this proposal are to determine the molecular mechanisms that modulate the efficiency and fidelity of 8-oxo-dG bypass in human cells, and ultimately the mutagenesis caused by oxidative DNA damage.
Exposure to environmental pollutants and other agents causes oxidative stress, a condition that underlies many disease states including cancer and the deleterious effects of aging. This research is designed to help elucidate the mechanism of how cells prevent mutations under conditions of oxidative stress. This will help us better understand specifically how exposure to such chemicals affects human health, and possibly to predict the affects of other agents that also cause oxidative stress.
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