PROJECT SUMMARY (See instructions): Excess reactive oxygen species, or oxidative stress, is a ubiquitous condition humans experience that damages the entire cell. Importantly, oxidative stress damages DNA resulting in numerous lesions that can halt DNA replication and increase mutagenesis. Oxidative stress emanates from various endogenous sources (metabolism, inflammation, etc.) but also exogenous environmental sources such as pollution, smoking, and ultraviolet radiation (UVR), arguably the most universal source of oxidative stress and DNA damage humans encounter. 8-oxodeoxyguaine (8oxoG) is a principle adduct generated by oxidative stress, and while well studied in vitro, is historically difficult to investigate in cells since the agents used to produce it (UVA, hydrogen peroxide, etc.) also generate other DNA lesions, and damage lipids and proteins in the cell. Our group developed and published on a novel fluorogen activated peptide (FAP) which can bind malachite green photosensitizer dyes and when excited with far-red light, specifically produces singlet oxygen. Singlet oxygen is known to have a short half-life and reacts rapidly with guanine to form 8oxoG. By fusing FAP to the telomere binding protein TRF1, we demonstrated the specificity of our chemoptogenetic system, and its spatial and temporal control. We also generated cells which express FAP fused to the histone H2B (H2B-FAP), allowing for genome-wide production of 8oxoG. The overall hypothesis of this proposal is that 8oxoG stalls DNA replication forks, especially at repetitive DNA sequences like telomeres, requiring the activities of ATR, Pol IJ, and PrimPol. This proposal is uniquely poised to address this hypothesis, as the H2B-FAP and TRF1-FAP tools are the only methods available to specifically induce 8oxoG within the human genome. In addition to telomeres, use of H2B-FAP will allow for the identification of other sequences sensitive to 8oxoG formation by examining the binding of replication stress response factors. Using physiological conditions, these identified sequences as well as telomere repeats will be studied in vitro to determine if they stall replicative DNA polymerases. This combination of biochemical and cellular replication studies will fill a critical gap in our knowledge of how 8oxoG impacts replication fork integrity and cell fate. Oxidative stress is linked to various diseases including cancer, but also aging. However, due to its pleiotropic effects, it is difficult to attribute any specific outcome to a particular lesion. While this study will advance our general understanding of 8oxoG, it will directly compare H2B-FAP activation with UVR, which induces pyrimidine dimers in addition to oxidative stress. UVR promotes skin carcinogenesis especially in the absence of factors like Pol 11, the protein mutated in the cancer predisposition syndrome, XPV. This study will examine the direct role of Pol 11 and other DNA replication factors (ATR, PrimPol, FANCD2, and MacroH2A1 .2) in the cellular response to 8oxoG.