Repair Mechanisms For Oxidative DNA Damage

DNA 氧化损伤的修复机制

• 批准号: 7325651
• 负责人: David M Wilson
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7325651
• 关键词: Repair; Mechanisms; Oxidative; DNA; Damage

To live, aerobic organisms metabolize oxygen to generate energy. During this process, cells create reactive oxygen species (ROS). ROS react with all cellular constituents, including lipids, proteins, and DNA. Such oxidative damage has been associated with the aging process and human disease, namely cancer and neurodegeneration. We have worked to define the biochemical and cellular processes for repairing oxidative DNA damage. In particular, we have delineated the structure-function mechanisms and biological contributions of specific proteins that participate in the base excision repair (BER) pathway. This process involves the recognition and excision of DNA damage, and restoration of the native genetic material. Defects in DNA repair give rise to mutations or cell death, leading to the development of disease. Much of our effort has involved defining the biochemical functions of Ape1, the major human protein for repairing abasic (AP) sites in DNA, a frequent genetic damage. We have demonstrated that Ape1 contributes to the repair of 3?-modifications in DNA as well, including mismatches, phosphate groups, phosphogycolates, and tyrosyl residues. Our more recent work has found that Ape1 cleaves at AP sites in single-stranded regions of complex, biologically-relevant DNA structures, such as bubble and fork intermediates. These findings expand the known repertoire of substrates processed by this enzyme, and suggest novel functions for Ape1 likely coupled to transcription and/or replication. Our recent work has focused on potential mechanisms of regulating Ape1 repair activities. For instance, we have demonstrated that the single-stranded DNA binding protein RPA inhibits promiscuous AP site incision by Ape1. In addition, CSB, a transcription-related repair protein defective in the human premature aging disorder Cockayne Syndrome, was found to activate Ape1 cleavage at AP sites in transcription bubble intermediates. Finally, our studies have discovered that the environmental metal, lead, is a potent inhibitor of Ape1 activity, suggesting a means by which this heavy metal may elicit its co-carcinogenic effects. We are currently designing methods to strategically regulate Ape1 repair activity in cells in the hopes of developing more effective anti-cancer treatment paradigms. In addition to the investigations above, we have initiated studies to determine the biochemical and cellular contributions of XRCC1, a major single-strand break repair (SSBR) factor. This protein functions primarily as a scaffold component, orchestrating specific protein-protein interactions required for efficient DNA repair. Recent work has identified associations of XRCC1 with proteins defective in human neurodegenerative disorders AOA1 (Aprataxin) and SCAN1 (TDP1). Our studies suggest a link of XRCC1 to replication via an interaction with PCNA, argue against a role for XRCC1 in the early steps of BER, and indicate a biologically-relevant role for its interaction with DNA polymerase beta and in the subsequent steps of SSBR, specifically DNA nick ligation. Ongoing studies using animal models (and derived cells) are determining the relationship of XRCC1 and oxidative DNA damage repair to aging and age-related disease, namely neurodegeneration. Additionally, we are determining the contribution, if any, of human XRCC1 and associated protein variants to impaired cellular responses that are related to disease manifestation.

为了生存，需氧生物代谢氧气以产生能量。在此过程中，细胞会产生活性氧 (ROS)。 ROS 与所有细胞成分发生反应，包括脂质、蛋白质和 DNA。这种氧化损伤与衰老过程和人类疾病（即癌症和神经退行性变）有关。我们致力于定义修复氧化 DNA 损伤的生化和细胞过程。特别是，我们描述了参与碱基切除修复（BER）途径的特定蛋白质的结构功能机制和生物学贡献。这个过程涉及DNA损伤的识别和切除，以及天然遗传物质的恢复。 DNA 修复缺陷会引起突变或细胞死亡，从而导致疾病的发生。 我们的大部分工作涉及定义 Ape1 的生化功能，Ape1 是修复 DNA 中脱碱基 (AP) 位点的主要人类蛋白质，这是一种常见的遗传损伤。我们已经证明，Ape1 也有助于修复 DNA 中的 3′-修饰，包括错配、磷酸基团、磷酸乙二醇酯和酪氨酰残基。我们最近的工作发现，Ape1 在复杂的、生物学相关的 DNA 结构（例如气泡和叉子中间体）的单链区域的 AP 位点上进行切割。这些发现扩展了该酶处理的已知底物库，并表明 Ape1 的新功能可能与转录和/或复制相关。我们最近的工作重点是调节 Ape1 修复活动的潜在机制。例如，我们已经证明单链 DNA 结合蛋白 RPA 可以抑制 Ape1 的 AP 位点混杂切割。此外，CSB（人类早衰症科凯恩综合症中存在缺陷的转录相关修复蛋白）被发现可以激活转录泡中间体中 AP 位点的 Ape1 裂解。最后，我们的研究发现环境金属铅是 Ape1 活性的有效抑制剂，这表明这种重金属可能引发其协同致癌作用。我们目前正在设计策略性调节细胞中 Ape1 修复活性的方法，希望开发出更有效的抗癌治疗范例。 除了上述研究之外，我们还启动了研究以确定 XRCC1（一种主要的单链断裂修复 (SSBR) 因子）的生化和细胞贡献。这种蛋白质的主要功能是作为支架成分，协调有效 DNA 修复所需的特定蛋白质-蛋白质相互作用。最近的工作已确定 XRCC1 与人类神经退行性疾病 AOA1 (Aprataxin) 和 SCAN1 (TDP1) 缺陷蛋白的关联。我们的研究表明 XRCC1 通过与 PCNA 相互作用与复制相关，反对 XRCC1 在 BER 早期步骤中的作用，并表明其与 DNA 聚合酶 beta 相互作用以及在 SSBR 的后续步骤（特别是 DNA 切口连接）中具有生物学相关的作用。正在进行的使用动物模型（和衍生细胞）的研究正在确定 XRCC1 和氧化 DNA 损伤修复与衰老和年龄相关疾病（即神经退行性疾病）的关系。此外，我们正在确定人类 XRCC1 和相关蛋白变体对与疾病表现相关的细胞反应受损的贡献（如果有的话）。