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，代谢副产品被称为活性氧（也称为自由基）。这些反应性产物攻击细胞成分，例如脂质，蛋白质和DNA。与DNA（即我们的遗传物质）的反应会导致几种损害中间体。如果未修复，这种损害会促进不良的遗传变化或导致细胞死亡。这样的终点与癌症，神经变性和衰老过程有关。为了调节这些结果，生物已经发展了一系列的修复系统，这些修复系统识别并去除了特定形式的DNA损伤。碱切除修复（BER）是修复氧化DNA损伤的主要途径，并涉及几种蛋白质的合作相互作用，这些蛋白会依次使用，以切除目标损伤并将DNA恢复为原始的未修饰形式。我们的重点是了解无碱性位点修复的分子机制和氧化DNA单链断裂。为此，我们已经隔离了几个BER蛋白参与者，并定义了他们的个体和合作结构 - 功能关系。我们的研究表明，APE1是AP位点的BER和主要哺乳动物修复蛋白的中心参与者，是一种特异性的核酸内切酶，它扫描DNA，具有与Abasic病变相关的独特柔韧性。我们已经定义了这种酶如何切割DNA（无碱性位点修复的第一步） - 一种催化反应机制，它很可能通过酶超家族在整个进化过程中得到保守。尽管APE1作为AP位点修复中的主要（如果不仅是）哺乳动物酶起作用，但我们已经表明，它在切除3'Blocking（例如磷酸盐）损害中具有更有针对性的作用，具体取决于DNA上下文/结构；因此，其他蛋白质可能会导致这种纠正过程。此外，我们已经证明APE1是去除某些3'末端不匹配的核苷酸，可能是诱变DNA中间体的编辑因素。我们目前正在使用生化，NMR光谱和晶体学技术来确定APE1与BER途径中其他蛋白质通信的机制，最著名的是DNA聚合酶β。我们的结构功能分析正在扩展到定义XRCC1的生化和细胞功能，XRCC1的生化和细胞功能是一种蛋白质，该蛋白质通过结合DNA迹象和间隙和募集BER蛋白来作为BER中的脚手架因子。涉及使用BLSA的临床样本的其他研究包括了解人口中发现的遗传变异对DNA修复功能的影响，假设某些遗传差异会产生蛋白质在DNA修复方面效率较低，从而使个人对疾病对疾病的影响更容易受到疾病或造成氧化损害的食物因素。总而言之，通过了解DNA修复的基本操作，我们正在建立一个基础，我们可以更好地了解氧化DNA损伤反应系统与人类疾病和衰老过程中遗传变异的关系。