Repair Mechanisms For Lesions And DNA Strand Breaks

损伤和 DNA 链断裂的修复机制

• 批准号: 6815329
• 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/6815329
• 关键词: DNA binding protein; DNA damage; DNA directed DNA polymerase; DNA repair; aging; endonuclease; genetic polymorphism; genetic susceptibility; human genetic material tag; longitudinal human study; nuclear magnetic resonance spectroscopy; oxidative stress; protein protein interaction

To live, humans convert oxygen to energy. During this process, metabolic byproducts are formed known as reactive oxygen species (also known as free radicals). These reactive products attack cellular constituents, such as lipids, proteins and DNA. Reactions with DNA, i.e. our genetic material, can lead to several damage intermediates. If unrepaired, this damage can promote unwanted genetic change or lead to cell death. Such end-points are associated with cancer, neurodegeneration, and the aging process. To regulate these outcomes, organisms have evolved an array of repair systems, which recognize and remove specific forms of DNA damage. Base excision repair (BER) is the major pathway for repairing oxidative DNA damage and involves the cooperative interaction of several proteins that work sequentially to excise the target damage and restore DNA back to its original, unmodified form. Our focus has been to understand the molecular mechanisms of repair of abasic sites and oxidative DNA single strand breaks. Towards this end, we have isolated several BER protein participants and are defining their individual and cooperative structure-function relationships. Our studies have revealed that Ape1, a central participant in BER and the major mammalian repair protein for AP sites, is a structure-specific endonuclease that scans DNA for a unique flexibility associated with the abasic lesion. We have defined how this enzyme cuts DNA - the first step in abasic site repair - a catalytic reaction mechanism that is likely conserved throughout evolution by a superfamily of enzymes. While Ape1 operates as the predominant (if not only) mammalian enzyme in AP site repair, we have shown that it has a more targeted role in the excision of 3'-blocking (e.g. phosphate) damages, depending on DNA context/structure; thus other proteins likely contribute to this corrective process. Moreover, we have shown that Ape1 is an editing factor that removes certain 3'-terminal mismatched nucleotides, potentially mutagenic DNA intermediates. We are presently determining the mechanism by which Ape1 communicates with other proteins in the BER pathway, most notably DNA polymerase beta, using biochemical, NMR spectroscopy and crystallography techniques. Our structure-function analysis is being expanded into defining the biochemical and cellular functions of Xrcc1, a protein that has been proposed to operate as a scaffolding factor in BER by binding DNA nicks and gaps, and recruiting BER proteins. Additional investigation, which involves the use of clinical samples from the BLSA, includes understanding the impact of genetic variation found in the human population on DNA repair function, with the hypothesis that certain genetic differences produce proteins that are less effective at DNA repair, thus rendering the individual more susceptible to disease upon exposure to environmental or food agents that create oxidative damage. In summary, by understanding the basic operations of DNA repair, we are building a foundation upon which we can better understand the relationship of genetic variation in oxidative DNA damage response systems to human disease and the aging process.

为了生存，人类将氧气转化为能量。在此过程中，代谢副产物形成称为活性氧物质（也称为自由基）。这些反应产物攻击细胞成分，如脂质、蛋白质和DNA。与DNA（即我们的遗传物质）的反应可能导致几种损害中间体。如果不修复，这种损伤可能会促进不必要的遗传变化或导致细胞死亡。这些终点与癌症、神经变性和衰老过程有关。为了调节这些结果，生物体已经进化出一系列修复系统，这些系统可以识别并消除特定形式的DNA损伤。碱基切除修复（BER）是修复氧化性DNA损伤的主要途径，涉及几种蛋白质的协同相互作用，这些蛋白质依次工作以切除靶损伤并将DNA恢复到其原始的未修饰形式。我们的重点是了解脱碱基位点和氧化DNA单链断裂修复的分子机制。为此，我们已经分离出几个BER蛋白的参与者，并正在定义他们的个人和合作的结构-功能关系。我们的研究表明，Ape 1是BER的核心参与者，也是AP位点的主要哺乳动物修复蛋白，它是一种结构特异性核酸内切酶，可以扫描DNA以获得与脱碱基损伤相关的独特灵活性。我们已经定义了这种酶如何切割DNA --无碱基位点修复的第一步--一种催化反应机制，可能在整个进化过程中被一个超家族的酶所保守。虽然Ape 1在AP位点修复中作为主要的（如果不是唯一的）哺乳动物酶，但我们已经证明它在切除3 '-阻断（例如磷酸盐）损伤中具有更有针对性的作用，这取决于DNA背景/结构;因此其他蛋白质可能有助于这种纠正过程。此外，我们已经证明Ape 1是一种编辑因子，可以去除某些3 '末端错配的核苷酸，潜在的致突变DNA中间体。我们目前正在确定Ape 1与BER途径中其他蛋白质（最值得注意的是DNA聚合酶β）通信的机制，使用生物化学，NMR光谱和晶体学技术。我们的结构-功能分析正在扩展到定义Xrcc 1的生物化学和细胞功能，Xrcc 1是一种蛋白质，已被提议通过结合DNA缺口和缺口并招募BER蛋白质来作为BER中的支架因子。其他研究涉及使用来自BLSA的临床样本，包括了解在人群中发现的遗传变异对DNA修复功能的影响，假设某些遗传差异产生的蛋白质在DNA修复方面效果较差，从而使个体在暴露于产生氧化损伤的环境或食物制剂时更容易患病。总之，通过了解DNA修复的基本操作，我们正在建立一个基础，在此基础上，我们可以更好地了解氧化DNA损伤反应系统中的遗传变异与人类疾病和衰老过程的关系。