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/文摘