Cross-links at abasic sites in duplex DNA

双链 DNA 脱碱基位点的交联

• 批准号: 10054954
• 负责人: Kent S Gates
• 金额: $ 39.78万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 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/10054954
• 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; Structure; 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

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.

项目摘要 内源性DNA损伤会导致衰老，零星癌和神经变性。有 内源性DNA损伤的许多来源，包括氧化，酗酒，核碱基脱氨酸和 糖苷键的水解产生脓肿部位。 DNA维修系统减轻了这些效果 内源性损害，但有些病变不可避免地逃避修复，并带来有害后果。重要的是， 并非所有的DNA损伤都是相等的。链间交联是异常的生物活性病变 阻止读出和复制细胞中遗传信息所需的DNA链分离。细胞 对其基因组中的交联答案没有好的答案：无法修复交联可能导致细胞死亡， 组织功能障碍和老化，而容易出错的修复可能会导致诱变和癌症。进化 并保留各行各业的精心交联维修系统 内源性链交联是不可避免的生活事实，但是这些交联的身份和 它们的生物后果仍然不确定。该应用的长期目标是评估 内源性DNA交联的发生和生物学终点，这将显着贡献 对癌症病因的总体理解。在上一个赠款期间的工作是一组 源自脓肿（AP）部位的链间交联，这是最常见的内源性病变 细胞DNA。这项工作进一步开发了用于检测和表征这些的LC-MS方法 双链DNA中的病变，并描述了一种用于评估效率和保真度的穿梭矢量方法 在人类细胞中修复并复制了AP衍生的交联。拟议的工作将（1） 进行无偏的屏幕以全面确定AP衍生的交联的序列热点 形成，（2）检测细胞DNA中AP衍生的交联的发生，（3）确定 在人类细胞中修复AP衍生的交联的效率和忠诚度并定义 在体外和人类细胞中依赖复制的交联的机制。拟议的工作 探讨以下假设：AP衍生的交联的形成和复制耦合修复可能是 在癌症，神经退行性和衰老中尤其重要。这项工作很重要，因为 内源性DNA交联的形成和修复可能有助于涉及的因果过程 衰老和早期癌变。这项工作具有创新性，因为它检查了结构新颖的交联链接 和新颖的维修机制，因此有望就内源性DNA损伤的作用进行新的见解 在人类健康和疾病中。从长远来看，结果可能使人们能够了解遗传 交联维修能力的差异有助于人类健康范围。最终这样的见解可以 激发通过遗传手段或药物来改善健康的方法 形成和/或增强内源交联的修复。