Cross-links at abasic sites in duplex DNA

项目总结 内源性DNA损伤可能导致衰老、散发性癌症和神经退化。确实有 内源性DNA损伤的多种来源，包括氧化、烷基化、碱基脱氨和 糖苷键的水解以产生碱性中心。DNA修复系统减轻了这些影响 内源性损伤，但一些损伤不可避免地逃避修复，造成有害后果。重要的是 并不是所有的DNA损伤都是平等的。链间交联物是一种特殊的生物活性损伤，因为它们 阻断在细胞中读出和复制遗传信息所需的DNA链分离。单元格 对它们基因组中的交联链没有很好的答案：不能修复交联链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。演变过程 各行各业对复杂的交叉链接修复系统的保留表明， 内源性链间交链是生命中不可避免的事实，但这些交链和 它们的生物学后果仍不确定。此应用程序的长期目标是评估 内源DNA交链的发生和生物终点，这将大大有助于 对癌症病因学有全面了解。前一批赠款期间的工作特点是一组 链间交叉链接源于基本(AP)部位，这是在 细胞DNA。这项工作进一步发展了LC-MS方法来检测和表征这些化合物 双链DNA中的损伤，并描述了一种评估穿梭载体方法的效率和保真度 AP衍生的交联链被修复并在人类细胞中复制。拟开展的工作，将(1) 进行无偏筛选，全面确定AP衍生交联链的序列热点 形成，(2)检测细胞DNA中AP衍生的交联链的出现，以及(3)确定 AP衍生的交叉连接在人类细胞中修复的效率和保真度，并定义了 在体外和在人类细胞中复制依赖的交联链“解钩”的机制。拟议中的工作 探索AP衍生的交叉链接的形成和复制耦合修复可能是 在癌症、神经退行性变和衰老中尤为重要。这项工作意义重大，因为 内源性DNA交联链的形成和修复可能有助于参与 衰老和早期致癌。这项工作具有创新性，因为它研究了结构新颖的交叉链接 和新的修复机制，从而对内源性DNA损伤的作用有了新的见解 在人类健康和疾病方面。从长远来看，这一结果可能有助于理解基因是如何 交链修复能力的差异有助于人类的健康。最终，这种洞察力可能会 启发通过遗传手段或通过抑制健康的药物来改善健康的方法 形成和/或增强内源性交联链的修复。

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.