Cross-links at abasic sites in duplex DNA

双链 DNA 脱碱基位点的交联

基本信息

批准号：
10295786
负责人：
Kent S Gates
金额：
$ 39.63万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-15 至 2023-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10295786
关键词：
Adenine Aging Aldehydes Alkylation Area BRCA1 gene Biological Biology Bypass Cancer Etiology Cell Death Cells Chemicals Chemistry Coupled DNA DNA Damage DNA Repair DNA Sequence Alteration DNA biosynthesis Deamination Dilution Techniques Disease Environmental Carcinogens Enzymes Evolution Exposure to Failure Functional disorder Generations Genetic Genetic Code Genetic Variation Genome Glycosides Grant Guanine Health Human Hydrolysis In Vitro Individual Inherited Laboratories Lead Lesion Life Light Malignant Neoplasms Methods Mutagenesis Mutation NEIL3 gene Nerve Degeneration Nucleic Acids Oligonucleotides Pharmacology Polymerase Predisposition Process Randomized Reaction Research Role Shuttle Vectors Site Source System Tissues Walking Work amino group carcinogenesis cigarette smoke crosslink detection method endonuclease genetic information healthspan improved innovation insight next generation sequencing novel nucleobase oxidation reconstitution repaired stable isotope

项目摘要

PROJECT SUMMARY Endogenous DNA damage can contribute to aging, sporadic cancers, and neurodegeneration. There are many sources of endogenous DNA damage, including oxidation, alkylation, nucleobase deamination, and hydrolysis of the glycosidic bond to generate abasic sites. DNA repair systems mitigate the effects of these endogenous damages, but some lesions inevitably evade repair with deleterious consequences. Importantly, not all DNA damage is created equal. Interstrand cross-links are exceptionally bioactive lesions because they block the DNA strand separation required for read-out and replication of genetic information in cells. Cells have no good answer to cross-links in their genome: Failure to repair cross-links may lead to cell death, tissue dysfunction, and aging, while error-prone repair may result in mutagenesis and cancer. The evolution and retention of elaborate cross-link repair systems across all walks of life suggest that the generation of endogenous interstrand cross-links is an unavoidable fact of life, but the identities of these cross-links and their biological consequences remain uncertain. The long-term objective of this application is to assess the occurrence and biological endpoints of endogenous DNA cross-links, which will contribute significantly to overall understanding of cancer etiology. Work during the previous grant period characterized a group of interstrand cross-links derived from an abasic (Ap) site that is the most common endogenous lesion found in cellular DNA. The work further developed LC-MS methods for the detection and characterization of these lesions in duplex DNA, and described a shuttle vector method for assessing the efficiency and fidelity with which the Ap-derived cross-links are repaired and replicated in human cells. The proposed work, will (1) conduct unbiased screens to comprehensively determine the sequence hotspots for Ap-derived cross-link formation, (2) detect the occurrence of Ap-derived cross-links in cellular DNA, and (3) determine the efficiency and fidelity with which Ap-derived cross-links are repaired in human cells and define the mechanisms of replication-dependent cross-link “unhooking” in vitro and in human cells. The proposed work explores the hypothesis that formation and replication-coupled repair of Ap-derived cross-links may be particularly important in cancer, neurodegeneration, and aging. The work is significant because the formation and repair of endogenous DNA cross-links may contribute to the causal processes involved in aging and early carcinogenesis. The work is innovative because it examines structurally novel cross-links and novel repair mechanisms, thus promising novel insights regarding the roles of endogenous DNA damage in human health and disease. In the long run, the results may enable understanding of how genetic differences in cross-link repair capacity contribute to human healthspan. Ultimately such insights could inspire approaches that improve health by genetic means or by pharmacological agents that inhibit the formation and/or enhance the repair of endogenous cross-links.

项目概要内源性 DNA 损伤可导致衰老、散发性癌症和神经退行性变。内源性 DNA 损伤的来源有很多，包括氧化、烷基化、核碱基脱氨和糖苷键水解产生脱碱基 DNA 修复系统可减轻这些影响。内源性损伤，但某些病变不可避免地无法修复，从而产生有害后果。并非所有的 DNA 损伤都是一样的，因为链间交联是具有特殊生物活性的损伤。阻断细胞内遗传信息读出和复制所需的 DNA 链分离。对于基因组中的交联没有好的答案：无法修复交联可能会导致细胞死亡，组织功能障碍和衰老，而容易出错的修复可能会导致突变和癌症。各行各业中复杂的交联修复系统的保留表明，内源性链间交联是生活中不可避免的事实，但这些交联的特性和它们的生物学后果仍不确定。该应用的长期目标是评估内源 DNA 交联的发生和生物学终点，这将显着有助于对癌症病因学的总体了解。源自脱碱基 (Ap) 位点的链间交联是最常见的内源性损伤这项工作进一步开发了用于检测和表征这些细胞的 LC-MS 方法。双链 DNA 中的损伤，并描述了一种穿梭载体方法，用于评估效率和保真度所提出的工作将 (1) 修复和复制 Ap 衍生的交联。进行无偏筛选，全面确定 Ap 衍生交联的序列热点形成，(2) 检测细胞 DNA 中 Ap 衍生交联的发生，以及 (3) 确定 Ap 衍生的交联在人体细胞中修复的效率和保真度，并定义了体外和人类细胞中复制依赖性交联“脱钩”的机制。探索了这样的假设：Ap 衍生的交联的形成和复制耦合修复可能是这项工作在癌症、神经退行性疾病和衰老方面尤其重要，因为内源 DNA 交联的形成和修复可能有助于参与的因果过程这项工作具有创新性，因为它研究了结构新颖的交联。和新颖的修复机制，从而有望对内源性 DNA 损伤的作用产生新的见解从长远来看，这些结果可能有助于了解遗传的影响。交联修复能力的差异最终可能有助于人类的健康寿命。激发通过遗传手段或通过抑制疾病的药物来改善健康的方法内源性交联的形成和/或增强内源性交联的修复。