Single Nucleotide Resolution Map of Formation and Repair of Bulky Adducts in the Human Genome

项目摘要/摘要 许多环境致癌物质和抗癌药物在基因组DNA中形成巨大的碱基加合物。 然而，这些损伤在整个人类基因组中的确切位置尚不清楚，而且 影响伤害形成和修复都很难用目前可用的方法来研究。因为 基因组的损伤和修复位置强烈影响病理条件的发生，有 需要新的方法来描绘整个基因组中的损伤和修复事件。长期的 我们研究计划的目标是更好地了解核苷酸切除修复系统是如何针对 从DNA中去除笨重的碱基加合物。这一特定提案的目标是进一步开发和应用 绘制致癌物和化疗诱导的DNA损伤形成和修复全程的新工具 并确定影响DNA损伤诱导和效率的关键因素 通过核苷酸切除修复来消除损伤。我们团队在DNA领域的背景和专业知识 修复酶学和基因组学使我们唯一有资格解决这个问题。对于这项提议，我们将 重点识别环境致癌物形成的DNA碱基损伤的准确位置 紫外光和苯并[a]芘以及以铂为基础的癌症化疗。我们最近开发了 独特的测序技术，我们称之为Damage-seq和xr-seq，可提供高分辨率DNA 分别关于整个人类基因组中损伤的形成和修复的序列信息。 这项拟议的研究的基本原理是，绘制损坏和修复地图的能力可能会揭示意想不到的 环境致癌物、诱变和特定基因组位置的人类疾病之间的联系 提出诊断和治疗人类癌症的新策略。我们对DNA加合物的基础研究 癌症风险和预防将在以下四个具体目标中进行审查：1)定量方法 绘制整个人类基因组的DNA损伤位置(损伤序列)图；2)定量方法 人类全基因组切除修复(XR-SEQ)图谱；3)全基因组加合物分析 形成和修复作为分化、细胞周期和染色质状态的函数；以及4)全基因组 生物有机体中加合物的形成和修复分析。我们最近提出的新方法 已开发并将进一步优化测绘，在整个工作中将使用损坏形成和修复。 这项提议是创新的，因为它为表征DNA损伤提供了一种新的和无与伦比的方法 由环境致癌物质和抗癌药物诱发。这项拟议的研究意义重大，因为它 有望极大地扩展我们对人类基因组DNA损伤形成和修复的理解 以前所未有的决心。归根结底，这些知识有可能改善预防 环境致癌战略，并导致开发诊断和治疗癌症的新工具 治疗人类癌症。

PROJECT SUMMARY/ABSTRACT Numerous environmental carcinogens and anti-cancer drugs form bulky base adducts in genomic DNA. However, the precise location of these lesions throughout the human genome is not known, and the factors that affect both damage formation and repair are difficult to study with currently available methodologies. Because the genomic location of damage and repair strongly influence the occurrence of pathological conditions, there is a need for new approaches for mapping damage and repair events across the entire genome. The long-term goal of our research program is to better understand how the nucleotide excision repair system targets the removal of bulky base adducts from DNA. The objective of this particular proposal is to further develop and apply novel tools for mapping carcinogen- and chemotherapy-induced DNA damage formation and repair throughout the genome and to identify the key factors that influence both the induction of DNA damage and the efficiency of damage removal by nucleotide excision repair. Our group’s background and expertise in the areas of DNA repair enzymology and genomics makes us uniquely qualified to address this issue. For this proposal, we will focus on identifying the precise locations of DNA base damage formed by the environmental carcinogens ultraviolet (UV) light and benzo[a]pyrene and by platinum-based cancer chemotherapies. We recently developed unique sequencing technologies that we have termed Damage-seq and XR-seq to provide high-resolution DNA sequence information on the formation and repair, respectively, of damage throughout the entire human genome. The rationale for the proposed research is that the ability to map damage and repair may reveal unexpected links between environmental carcinogens, mutagenesis, and human disease at specific genomic sites and suggest new strategies for diagnosing and treating human cancers. Our basic research on DNA adducts in cancer risk and prevention will be examined in the following four specific aims: 1) Method for Quantitative Mapping of DNA Damage Sites (Damage-seq) across the Whole Human Genome; 2) Method for Quantitative Mapping of Excision Repair (XR-seq) of the Whole Human Genome; 3) Genome-wide Analysis of Adduct Formation and Repair as a Function of Differentiation, Cell Cycle, and Chromatin States; and 4) Genome-wide Analysis of Adduct Formation and Repair in Human Biospecimens. The novel methods that we recently developed and will further optimize for mapping damage formation and repair will be used throughout this work. This proposal is innovative because it provides a new and unparalleled approach for characterizing DNA damage induced by environmental carcinogens and anti-cancer drugs. The proposed research is significant because it is expected to significantly expand our understanding of DNA damage formation and repair in the human genome at an unprecedented level of resolution. Ultimately, this knowledge has the potential to improve the prevention strategies for environmental carcinogenesis and to lead to the development of new tools for diagnosing and treating human cancer.