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

研究 8-氧代鸟嘌呤对 DNA 复制和基因组稳定性的细胞影响

• 批准号: 10348923
• 负责人: Ryan P Barnes
• 金额: $ 10.72万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-06 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10348923
• 关键词: Address; Aging; Aspergillus Nuclease S1; Base Excision Repairs; Binding; Biochemical; Biochemistry; Biological; Bypass; Cell Aging; Cell Death; Cell Line; Cell Survival; Cells; ChIP-seq; Chimeric Proteins; Chromatin; Chromosome Fragile Sites; Comb animal structure; Complement; DNA; DNA Adducts; DNA Damage; DNA Replication Factor; DNA Sequence; DNA analysis; DNA biosynthesis; DNA photoproducts; DNA replication fork; DNA-Directed DNA Polymerase; Disease; Dyes; FANCD2 protein; Fiber; Gamma-H2AX; Genome; Genome Stability; Genomic DNA; Genomic Instability; Genomics; Guanine; Half-Life; Histone H2B; Histones; Human; Human Genome; Hydrogen Peroxide; In Vitro; Inflammation; Knock-out; Knowledge; Lesion; Light; Link; Lipids; Malignant Neoplasms; Mediating; Metabolism; Methods; Mitosis; Mitotic; Mutagenesis; Mutate; Mutation; Nitrogen; Nuclear; OGG1 gene; Outcome; Oxidants; Oxidative Stress; Oxygen; Pathway interactions; Peptides; Photosensitizing Agents; Physiological; Pollution; Polymerase; Production; Proteins; Publishing; Pyrimidine Dimers; Regulation; Reporter; Role; Singlet Oxygen; Site; Skin Carcinogenesis; Smoking; Solar Energy; Source; Specificity; Stress; Syndrome; System; TERF1 gene; Telomere-Binding Proteins; Telomeric Repeat Binding Protein 1; Transfection; UVA induced; Ultraviolet Rays; adduct; base; biological adaptation to stress; cancer cell; cancer predisposition; cell transformation; chromophore; experience; experimental study; genome sequencing; genome-wide; human DNA damage; irradiation; mutant; novel; oxidative DNA damage; pleiotropism; prevent; repaired; replication stress; response; senescence; solar ultraviolet radiation; telomere; tool; whole genome

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.

项目概要/摘要 过量的活性氧/氮或氧化应激是人类普遍存在的情况 会损害整个细胞。重要的是，氧化应激会损害 DNA，导致许多损伤， 停止 DNA 复制并增加诱变。氧化应激来自各种内源性来源 （新陈代谢、炎症等）以及外源性环境来源，例如污染、吸烟和 太阳紫外线辐射 (UVR)，可以说是最普遍的氧化应激和 DNA 损伤来源 人类相遇。 8-氧代-脱氧鸟嘌呤 (8oxoG) 是氧化应激产生的主要加合物之一， 虽然在体外进行了充分研究，但历史上很难在细胞中进行研究，因为用于生产它的试剂 （UVA、过氧化氢等）还会产生其他 DNA 加合物、链断裂，并损害脂质和蛋白质 整个细胞。我们的团队开发并发表了一种新型荧光激活肽（FAP）， 可以结合孔雀石绿光敏剂染料，当用远红光激发时，特异性地产生单线态 氧。已知单线态氧的半衰期很短，并且会与鸟嘌呤快速反应形成 8oxoG。经过 将 FAP 与端粒结合蛋白 TRF1 融合，我们能够证明我们的特异性 化学光遗传学系统及其空间和时间控制。我们还生成了表达的细胞系 FAP 与组蛋白 H2B (H2B-FAP) 融合，允许在全基因组范围内生产 8oxoG。总体假设 该提议的主要内容是，8oxoG 会阻止 DNA 复制叉，尤其是在端粒等重复 DNA 序列处， 需要 ATR、Pol η 和 PrimPol 的活性。该提案旨在解决这一假设， 因为 H2B-FAP 和 TRF1-FAP 工具是唯一可在细胞内特异性诱导 8oxoG 的方法。 人类基因组。除了端粒之外，使用 H2B-FAP 工具还可以识别其他端粒 通过 ChIP-检查复制应激反应因子的结合来确定对 8oxoG 形成敏感的序列 序列。使用生理条件，将研究这些已识别的序列以及端粒重复序列 体外以确定它们是否阻碍复制 DNA 聚合酶。这种生物化学和细胞的结合 复制研究将填补我们关于 8oxoG 如何影响复制叉完整性的知识空白 和细胞命运。氧化应激与多种疾病有关，包括癌症，但也与衰老有关。然而，由于 由于其多效性效应，很难将任何特定结果归因于特定病变。虽然这项研究将 推进我们对 8oxoG 的一般理解，它将直接比较 H2B-FAP 激活与 UVA（一种特定的 UVR 的子集），除了氧化应激外，它还会诱导嘧啶二聚体。 UVR促进皮肤 致癌作用，尤其是在缺乏 Pol η（癌​​症中突变的蛋白质）等因素的情况下 易感综合征，XPV。这项研究还将检验 Pol η 和其他 DNA 复制的直接作用 细胞对 8oxoG 反应的因素（ATR、PrimPol、FANCD2 和 MacroH2A1.2）。