DNA Adduct Detection and Repair in Mammalian Cells

项目摘要/摘要 许多内源性物质、环境致癌物和抗癌药物会产生大量的碱基加合物 在基因组中。预计这些加合物的位置是非随机的，并且它们的位置决定了 它们的致病或治疗作用以及它们对调节这些基因的DNA修复酶的敏感性 效果。我们研究计划的长期目标是更好地了解DNA损伤和修复 基因组水平，以帮助预测和潜在地预防DNA加合物诱导的致癌以及 设计副作用最小的高效化疗方案。这项特别提案的目标是 使用我们的新的加合映射方法(Damage-Seq和XR-Seq)来定位DNA的准确位置 核苷酸切除对哺乳动物基因组的损伤和DNA切开的确切位置 修复酶系统，消除DNA损伤。这项拟议研究的基本原理是 损伤和修复可能揭示环境致癌物、诱变、疾病和 老龄化。我们将通过实现以下三个具体目标来实现这一目标：1)基因组单- DNA内源性损伤的核苷酸拆分分析2)全基因组单核苷酸 外源致癌物对DNA损伤和修复的分辨图谱；3)全基因组损伤和修复 抗癌药物地图。对于这项建议，我们将重点确定准确的位置和拆除 DNA碱基损伤内源性形成(糖基化、氧化损伤) 环境致癌物质(黄曲霉毒素、二乙基亚硝胺)和抗癌药物(顺铂、马法兰)。方法 我们开发并进一步优化的地图伤害形成和修复将在整个过程中使用 工作。这项提议是创新的，因为这些独特的测序技术提供了高分辨率 关于整个基因组中损伤的形成和修复的DNA序列信息，并提供 无与伦比的方法表征内源性DNA损伤以及由 环境致癌物质和抗癌药物。拟议的研究具有重要意义，因为它将解决 不同组织的内源性因素(糖基化)对DNA损伤的作用及其整体 我们实验室开发的这些方法对衰老的贡献具有前所未有的敏感性 在不同器官中以单核苷酸分辨率绘制全基因组图谱。此外，拟议的研究将 以前所未有的速度扩大我们对人类基因组中DNA损伤形成和修复的理解 关于内源性和由环境致癌物和环境致癌物和 抗癌药物。归根结底，这些知识有可能改进预防策略 环境致癌，并导致诊断和治疗癌症的新工具的开发。

PROJECT SUMMARY/ABSTRACT Numerous endogenous agents, environmental carcinogens, and anti-cancer drugs produce bulky base adducts in the genome. It is expected that the location of these adducts is non-random and that their locations dictate their pathogenic or therapeutic effects as well as their susceptibility to DNA repair enzymes that modulate these effects. The long-term goal of our research program is to better understand DNA damage and repair at the genomic level to aid in predicting and potentially preventing DNA adduct-induced carcinogenesis as well as to design efficient chemotherapeutic regimens with minimal side effects. The objective of this particular proposal is to use our novel adductomic-mapping methods (Damage-seq and XR-seq) to locate the exact positions of DNA lesions in the mammalian genome and the exact positions of DNA incisions performed by the nucleotide excision repair enzyme system that removes the DNA damage. The rationale for the proposed research is that mapping damage and repair may reveal unexpected links between environmental carcinogens, mutagenesis, disease and ageing. We will accomplish this goal by carrying out the following three specific aims: 1) Genomic Single- nucleotide Resolution Analysis of DNA Damage by Endogenous Agents; 2) Genome-wide Single Nucleotide Resolution Maps of DNA Damage and Repair by Exogenous Carcinogens; 3) Genome-wide Damage and Repair Maps of Anticancer Drugs. For this proposal, we will focus on identifying the precise locations and removal of DNA base damage formed endogenously (glycosylation, oxidative damage) and damage caused by environmental carcinogens (aflatoxin, diethylnitrosamine) and anticancer drugs (cisplatin, melphalan). Methods that we developed and further optimized for mapping damage formation and repair will be used throughout this work. This proposal is innovative because of these unique sequencing technologies that provide high-resolution DNA sequence information on the formation and repair of damage throughout the entire genome and provide an unparalleled approach for characterizing endogenous DNA damage as well as damage induced by environmental carcinogens and anti-cancer drugs. The proposed research is significant because it will address the question of the role of DNA damage by endogenous agents (glycosylation) of various tissues and its overall contribution to aging by these methods developed in our laboratory which have unprecedented sensitivity for genome-wide mapping at single nucleotide resolution in different organs. In addition, the proposed research will expand our understanding of DNA damage formation and repair in the human genome at an unprecedented level of detail regarding genomic damage formed endogenously and by environmental carcinogens and anticancer drugs. 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 cancer.