DNA–Protein Cross-Linking Sequencing for Genome-Wide Mapping of Abasic Sites at Single-Nucleotide Resolution

DNA-蛋白质交联测序，以单核苷酸分辨率进行全基因组脱碱基位点作图

• 批准号: 10723069
• 负责人: Feng Tang
• 金额: $ 11.91万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-11 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723069
• 关键词: Ablation; Acceleration; Affect; Age; Aging; Alkylation; Animal Model; Applications Grants; B-Lymphocytes; Bioinformatics; CRISPR/Cas technology; Cancer Etiology; Cells; Chromatin; Cytolysis; DNA; DNA Damage; DNA Repair; DNA glycosylase; DNA lesion; DNA-(apurinic or apyrimidinic site) lyase; DNA-Directed DNA Polymerase; DNA-protein crosslink; Data; Deamination; Depurination; Disease; Environmental Exposure; Enzymes; Epigenetic Process; Etiology; Failure; Generations; Genes; Genetic; Genome; Genome Mappings; Genomic DNA; Genomic Instability; Genomics; Glycols; Goals; Histones; Human; Human Genome; Hydrolysis; Knock-out; Knowledge; Learning; Malignant Neoplasms; Maps; Mediating; Mentors; Metabolism; Methods; Mutagenesis; Mutation; Mutation Analysis; Neurodegenerative Disorders; Nucleotides; Outcome; Pattern; Prevention strategy; Process; Publications; Publishing; Research; Research Project Grants; Resolution; Role; Scheme; Scientist; Site; Source; Testing; Therapeutic Intervention; Thymidine; Toxicant exposure; Toxicology; Training; Wild Type Mouse; Work; base; cancer genome; career; environmental toxicology; experience; genome sequencing; genome-wide; human disease; innovation; insight; mouse model; next generation sequencing; novel; oxidation; preventive intervention; repaired; skills; toxicant; whole genome

Project Summary Endogenous metabolism and environmental exposure can result in a battery of DNA lesions. Failure to repair these DNA lesions gives rise to genome instability and mutation accrual, which lead to accelerated aging, cancer, and neurodegenerative diseases. Abasic site (AP site) is one of the most prevalent forms of DNA damage. Although AP sites are known to be repaired by AP endonuclease in human cells, the dynamics of AP site induction and repair across the genome and the cellular factors modulating these processes remain elusive. In this application, we aim to explore how site-specific induction and repair of AP sites are affected by toxicant exposure and epigenetic alterations. We propose three specific aims to achieve our objective: 1) we will develop a DNA-protein cross-linking followed by next-generation sequencing (DPC-Seq) method for mapping, at single- base resolution, AP sites in the human genome after toxicant exposure; 2) we will explore how the formation and repair of AP sites are influenced by epigenetic state of chromatin; and 3) we will employ DPC-seq and whole- genome mutation analysis to examine how AP sites contribute to mutational signatures observed in cancer genomes. To accomplish this proposal and enable my transition to an independent academic career, I assembled a mentoring committee with expertise in animal models, bioinformatics, mutagenesis, and toxicology. The proposed research is built upon my research experience/skill sets, strong preliminary data, and the proposed mentoring plan. The outcome of the proposed research will establish a new method for mapping AP sites at single-base resolution and provide important insights into the occurrence and repair of AP sites and how they are influenced by toxicant exposure and genomic contexts. The proposed research will also reveal the roles of AP sites in cancer etiology, which will ultimately lead to better strategies for the prevention and therapeutic interventions of human cancer. Moreover, this proposal will fill gaps in my training and collect the data for my independent publications and research grant applications, thereby enabling me to transition to become an independent scientist in the field of environmental toxicology.

项目概要 内源性代谢和环境暴露可能导致一系列 DNA 损伤。无法修复 这些 DNA 损伤会导致基因组不稳定和突变增加，从而导致加速衰老、癌症、 和神经退行性疾病。无碱基位点（AP 位点）是最常见的 DNA 损伤形式之一。 尽管已知人类细胞中的 AP 位点可被 AP 核酸内切酶修复，但 AP 位点的动态变化 整个基因组的诱导和修复以及调节这些过程的细胞因子仍然难以捉摸。 在此应用中，我们旨在探索有毒物质如何影响 AP 位点的位点特异性诱导和修复 暴露和表观遗传改变。我们提出三个具体目标来实现我们的目标：1）我们将开发 DNA-蛋白质交联，然后采用下一代测序 (DPC-Seq) 方法进行绘图，在单 碱基分辨率，有毒物质暴露后人类基因组中的 AP 位点； 2）我们将探讨如何形成和 AP位点的修复受到染色质表观遗传状态的影响； 3）我们将采用DPC-seq和whole- 基因组突变分析，检查 AP 位点如何影响癌症中观察到的突变特征 基因组。为了完成这个提案并使我能够过渡到独立的学术生涯，我 组建了一个具有动物模型、生物信息学、诱变和毒理学专业知识的指导委员会。 拟议的研究是建立在我的研究经验/技能、强有力的初步数据和拟议的研究基础上的 辅导计划。 拟议研究的结果将建立一种在单碱基上绘制 AP 位点的新方法 解决方案并提供有关 AP 位点的发生和修复以及它们如何受到影响的重要见解 通过有毒物质暴露和基因组背景。拟议的研究还将揭示 AP 位点在 癌症病因学，最终将导致更好的预防和治疗干预策略 人类癌症。此外，该提案将填补我的培训空白并为我的独立收集数据 出版物和研究资助申请，从而使我能够过渡为独立的 环境毒理学领域的科学家。