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.

项目总结/摘要 过量的活性氧/氮物质或氧化应激是人类普遍经历的一种情况， 会破坏整个细胞。重要的是，氧化应激会损害DNA，导致许多病变， 阻止DNA复制并增加诱变。氧化应激来自各种内源性来源 （代谢、炎症等）还有外源性环境源，如污染、吸烟， 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 人类相遇8-氧代-脱氧胍（8 oxoG）是由氧化应激产生的主要加合物之一， 虽然在体外研究得很好，但历史上很难在细胞中进行研究，因为用于产生它的试剂 (UVA、过氧化氢等）也会产生其他DNA加合物、链断裂，并破坏脂质和蛋白质 整个细胞。我们的小组已经开发并发表了一种新的荧光激活肽（FAP）， 可结合孔雀石绿色光敏染料，当用远红光激发时， 氧气已知单线态氧具有短半衰期，并且与鸟嘌呤快速反应形成8 oxoG。通过 将FAP与端粒结合蛋白TRF 1融合，我们能够证明我们的特异性。 化学光遗传系统及其时空调控。我们还建立了表达 FAP融合到组蛋白H2 B（H2 B-FAP），允许全基因组产生8 oxoG。总体假设 这个提议的一个重要原因是，8 oxoG阻止了DNA复制叉，特别是在端粒等重复DNA序列上， 需要ATR、Pol η和PrimPol的活性。这一建议是唯一准备解决这一假设， 由于H2 B-FAP和TRF 1-FAP工具是唯一可用于特异性诱导8 oxoG的方法， 人类基因组除了端粒，使用H2 B-FAP工具将允许识别其他端粒。 序列敏感的8 oxoG形成通过检查复制应激反应因子的结合，通过ChIP- seq.使用生理条件，这些鉴定的序列以及端粒重复序列将在 以确定它们是否能阻止复制型DNA聚合酶。这种生物化学和细胞学的结合 复制研究将填补我们对8 oxoG如何影响复制叉完整性的知识的关键空白 细胞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。但由于 由于其多效性，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 推进我们对8 oxoG的一般理解，它将直接比较H2 B-FAP激活与UVA（一种特异性的 UVR的子集），其除了氧化应激之外还诱导嘧啶二聚体。紫外线促进皮肤 致癌作用，特别是在缺乏诸如Pol η（癌症中突变的蛋白质）等因素的情况下 易感综合征本研究还将研究Pol η和其他DNA复制的直接作用 在细胞对8 oxoG的反应中，FANCD 2和MacroH2A1.2是细胞因子（ATR、PrimPol、FANCD 2和MacroH2A1.2）的主要作用。