DNA Adduct Detection and Repair in Mammalian Cells

项目总结/摘要 许多内源性物质、环境致癌物和抗癌药物都能产生大体积的碱加合物 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作用的DNA修复酶的易感性， 方面的影响.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加合物诱导的致癌作用，以及 设计副作用最小的有效化疗方案。这项建议的目的是 使用我们新的内收酶标测方法（Damage-seq和XR-seq）来定位DNA的确切位置， 哺乳动物基因组中的损伤和核苷酸切除所进行的DNA切口的确切位置 修复酶系统，消除DNA损伤。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- 内源性物质对DNA损伤的核苷酸分辨率分析; 2）全基因组单核苷酸 外源性致癌物引起的DNA损伤和修复的分辨率图; 3）全基因组损伤和修复 抗癌药物地图。就这项建议而言，我们会集中研究如何确定 内源性DNA碱基损伤（糖基化、氧化损伤）和 环境致癌物（黄曲霉毒素、二乙基亚硝胺）和抗癌药物（顺铂、美法仑）。方法 我们开发并进一步优化的映射损伤形成和修复将在整个过程中使用， 工作这项建议是创新的，因为这些独特的测序技术，提供高分辨率 DNA序列信息的形成和修复损伤的整个基因组，并提供了一个 这是一种无与伦比的方法，用于表征内源性DNA损伤以及 环境致癌物和抗癌药物。这项研究意义重大，因为它将解决 各种组织的内源性因子（糖基化）对DNA损伤的作用及其总体影响的问题， 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灵敏度， 在不同器官中以单核苷酸分辨率进行全基因组作图。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 关于内源性和环境致癌物形成的基因组损伤的详细程度， 抗癌药最终，这些知识有可能改善预防战略， 环境致癌作用，并导致诊断和治疗癌症的新工具的发展。

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.