Cross-links at abasic sites in duplex DNA

项目概要 内源性 DNA 损伤可能导致衰老、散发性癌症和神经退行性变。有 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位点。 DNA 修复系统可减轻这些影响 内源性损伤，但有些病变不可避免地无法修复，从而产生有害后果。重要的是， 并非所有 DNA 损伤都是一样的。链间交联是异常具有生物活性的损伤，因为它们 阻断细胞内遗传信息读出和复制所需的 DNA 链分离。细胞 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导致突变和癌症。演变 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。该应用程序的长期目标是评估 内源 DNA 交联的发生和生物学终点，这将显着有助于 对癌症病因学的全面了解。上一个资助期间的工作特点是一群 源自脱碱基 (Ap) 位点的链间交联是最常见的内源性损伤 细胞DNA。这项工作进一步开发了用于检测和表征这些物质的 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 其中Ap衍生的交联在人体细胞中被修复和复制。拟议的工作将 (1) 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。拟议的工作 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 在癌症、神经退行性疾病和衰老中尤其重要。这项工作意义重大，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 衰老和早期癌变。这项工作具有创新性，因为它研究了结构新颖的交联 和新的修复机制，从而有望对内源性 DNA 损伤的作用有新的见解 在人类健康和疾病中。从长远来看，这些结果可能有助于了解遗传是如何发生的。 交联修复能力的差异有助于人类的健康寿命。最终这些见解可以 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。

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.