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，有氧生物会代谢氧气产生能量。在此过程中，细胞会产生活性氧（ROS）。 ROS与包括脂质，蛋白质和DNA在内的所有细胞成分反应。这种氧化损伤与衰老过程和人类疾病有关，即癌症和神经退行性。我们努力定义用于修复氧化DNA损伤的生化和细胞过程。特别是，我们描述了参与基础切除修复（BER）途径的特定蛋白质的结构功能机制和生物学贡献。该过程涉及对DNA损伤的识别和切除，以及对天然遗传物质的恢复。 DNA修复中的缺陷导致突变或细胞死亡，导致疾病的发展。 我们的大部分努力都涉及定义APE1的生化功能，APE1是修复DNA中无碱（AP）位点的主要人蛋白，这是一种频繁的遗传损害。我们已经证明，APE1也有助于修复DNA中的3次ape1，包括错配，磷酸基团，磷酸盐和酪酶残基。我们最近的工作发现，APE1在复杂的，生物学上与生物学的DNA结构（例如气泡和叉子中间体）的单链区域的AP位点切割。这些发现扩大了该酶处理的底物的已知曲目，并提出了可能与转录和/或复制结合的APE1的新功能。我们最近的工作集中在调节APE1维修活动的潜在机制上。例如，我们已经证明了单链DNA结合蛋白RPA抑制APE1的混杂AP位点切口。此外，发现与转录相关的修复蛋白有缺陷的CSB在人体过早衰老疾病中有缺陷，可在转录气泡中间体中激活APEC的APE1裂解。最后，我们的研究发现，环境金属铅是一种有效的APE1活性抑制剂，这表明这种重金属可能会引起其共癌作用。目前，我们正在设计方法来策略性地调节细胞中的APE1修复活性，以期开发更有效的抗癌治疗范例。 除了上面的研究外，我们还开始研究以确定XRCC1的生化和细胞贡献，XRCC1是主要的单链断裂修复（SSBR）因子。该蛋白质主要是脚手架成分，策划了有效的DNA修复所需的特定蛋白质 - 蛋白质相互作用。最近的工作已经确定了XRCC1与人神经退行性疾病中有缺陷的AOA1（Aprataxin）和Scan1（TDP1）中有缺陷的蛋白的关联。我们的研究表明，XRCC1通过与PCNA的相互作用进行了复制的联系，反对XRCC1在BER的早期步骤中的作用，并指示其与DNA聚合酶β的相互作用以及在SSBR的后续步骤中的相互作用，特定于DNA Nick结扎。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损伤修复与衰老和与年龄有关的疾病的关系，即神经变性。此外，我们正在确定人XRCC1和相关蛋白质变异对与疾病表现相关的细胞反应的贡献（如果有的话）。