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: 10534764
• 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 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10534764
• 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; Complement; DNA; DNA Adduction; DNA Adducts; DNA Damage; DNA Replication Factor; DNA Sequence; DNA analysis; DNA biosynthesis; DNA photoproducts; DNA replication fork; DNA-Directed DNA Polymerase; Disease; Dyes; Environment; 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; Reaction; Regulation; Reporter; Role; Single-Stranded DNA; 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; macroH2A histone; micronucleus; 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.

项目摘要/摘要 过量的活性氧/氮或氧化物胁迫是人类经历的一种无处不在的疾病 会损坏整个牢房。重要的是，氧化应激会损害DNA，导致许多病变 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，感染等），但还包括污染，吸烟和 太阳紫外线辐射（UVR），可以说是氧化应激和DNA损伤的最普遍的来源 人类遇到。 8-oxo-deoxyGuaine（8oxog）是由氧化应激产生的原理加成之一， 尽管在体外进行了良好的研究，但历史上很难在细胞中进行研究，因为用来生产它的药物 （UVA，过氧化氢等）还会产生其他DNA加合物，链断裂，损伤脂质和蛋白质 通过细胞。我们的小组已经开发并发表了一种新型的荧光激活肽（FAP），该肽（FAP） 可以绑定孔雀石绿色光敏剂染料，并在用远红色的光激发时，专门产生单线 氧。已知单线氧的半衰期短，并与鸟嘌呤迅速反应形成8oxog。经过 将FAP融合到端粒结合蛋白TRF1上，我们能够证明我们的特异性 化学发生系统及其空间和临时控制。我们还生成了表达的细胞系 FAP融合了组蛋白H2B（H2B-FAP），从而允许全基因组的8oxog产生。总体假设 该建议的是，8oxog失速DNA复制叉，尤其是在端粒等重复的DNA序列中， 需要ATR，Polη和Primpol的活动。该提议是独特的，以解决这一假设， 由于H2B-FAP和TRF1-FAP工具是唯一可以专门诱导8oxog的方法 人基因组。除端粒外，使用H2B-FAP工具还可以识别其他 序列通过芯片检查复制应力响应因子的结合，对8oxog的形成敏感 seq。使用物理条件，这些确定的序列以及端粒重复序列将在 体外以确定它们是否停滞复制性DNA聚合酶。生化和细胞的这种组合 复制研究将填补我们对8oxog如何影响复制叉完整性如何影响的差距 和细胞命运。氧化应激与包括癌症以及衰老在内的各种疾病有关。但是，由于 它的多效效应，很难将任何特定结果归因于特定病变。虽然这项研究将 促进我们对8oxog的一般理解，它将直接将H2B-FAP激活与UVA进行比较（一个特定 UVR的子集，除氧化应激外，还诱导嘧啶二聚体。 UVR促进皮肤 致癌作用，尤其是在没有诸如polη之类的因素的情况下，蛋白在癌症中突变 倾向综合征，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的细胞反应中的因素（ATR，Primpol，Fancd2和MacroH2A1.2）。