Investigating the Cellular Impact of 8-oxo-Guanine on DNA Replication and Genome Stability

项目摘要/摘要 过量的活性氧/氮物种，或氧化应激，是人类经历的一种普遍情况 会破坏整个细胞。重要的是，氧化应激损伤DNA，导致大量损伤，从而 阻止DNA复制并增加突变。氧化应激来自各种内源性来源 (新陈代谢、炎症等)而且还有外源环境源，如污染、吸烟和 太阳紫外线辐射(UVR)，可以说是氧化应激和DNA损伤的最普遍来源 人类遇到了。8-氧代脱氧鸟嘌呤(8oxoG)是氧化应激产生的主要加合物之一， 虽然在体外进行了很好的研究，但历史上很难在细胞中进行研究，因为生产它的试剂 (UVA、过氧化氢等)还会产生其他DNA加合物、链断裂，并损害脂类和蛋白质 遍及整个细胞。我们团队已经开发并发表了一种新型的氟激活肽(FAP)，它可以 能结合孔雀石绿光敏剂染料，在远红光激发下，专门产生单线态 氧气。众所周知，单线态氧的半衰期很短，并能迅速与鸟嘌呤反应生成8oxoG。通过 将FAP与端粒结合蛋白TRF1融合，我们能够证明我们的 化学发生系统及其时空控制。我们还培育出了表达基因的细胞系 FAP与组蛋白H_2B(H_2B-FAP)融合，允许全基因组生产8oxoG。总体假设 这一提议的核心是，8oxoG会拖延DNA复制分叉，特别是在端粒等重复DNA序列上， 需要ATR、POLη和PRIMPOL的活动。这一提议是唯一能够解决这一假设的方案， 由于H2B-FAP和TRF1-FAP工具是仅有的在体内特异性诱导8oxoG的方法 人类基因组。除了端粒，使用H2B-FAP工具还可以识别其他 通过芯片检测复制应激反应因子的结合来检测对8oxoG形成敏感的序列 序列号。利用生理条件，这些已识别的序列以及端粒重复将在 以确定它们是否阻碍了复制的DNA聚合酶。这种生化和细胞的结合 复制研究将填补我们关于8oxoG如何影响复制分叉完整性的知识的一个关键空白 和细胞的命运。氧化应激与包括癌症在内的各种疾病有关，但也与衰老有关。然而，由于 由于它的多效性，很难将任何特定的结果归因于特定的病变。虽然这项研究将 增进我们对8oxoG的总体理解，它将直接比较H_2B-FAP和UVA(一种特异性的 UVR的子集)，除了氧化应激外，它还诱导嘧啶二聚体。UVR促进皮肤 致癌，特别是在缺乏POLη等因素的情况下，这种蛋白质在癌症中发生突变 易感综合征，XPV。这项研究还将检查POLη和其他DNA复制的直接作用 细胞对8oxoG反应中的因子(ATR、PrimPol、FANCD2和MacroH2A1.2)。

Project Summary/Abstract Excess reactive oxygen/nitrogen species, or oxidative stress, is a ubiquitous condition humans experience that can damage 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 solar ultraviolet radiation (UVR), arguably the most universal source of oxidative stress and DNA damage humans encounter. 8-oxo-deoxyguaine (8oxoG) is one of the principle adducts 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 adducts, strand-breaks, and damage lipids and proteins throughout the cell. Our group has 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 were able to demonstrate the specificity of our chemoptogenetic system, and its spatial and temporal control. We have also generated cell lines 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 η, 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 the H2B-FAP tool will allow for the identification of other sequences sensitive to 8oxoG formation by examining the binding of replication stress response factors by ChIP- seq. 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 UVA (a specific subset of UVR), which induces pyrimidine dimers in addition to oxidative stress. UVR promotes skin carcinogenesis especially in the absence of factors such as Pol η, the protein mutated in the cancer predisposition syndrome, XPV. This study will also examine the direct role of Pol η and other DNA replication factors (ATR, PrimPol, FANCD2, and MacroH2A1.2) in the cellular response to 8oxoG.