Repair Mechanisms For Strand Breaks in DNA

DNA 链断裂的修复机制

• 批准号: 6668151
• 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/6668151
• 关键词: DNA damage; DNA repair; endonuclease; genetic polymorphism; genetic susceptibility; nuclear magnetic resonance spectroscopy; oxidative stress; protein structure function

To live, humans convert oxygen to energy. Yet during this process, metabolic byproducts are formed known collectively as reactive oxygen species (also known as free radicals). These reactive products attack various cellular constituents, including 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 human disease, most notably cancer and neurodegeneration, and to 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 the DNA back to its original, unmodified form. In brief, the main steps of BER consist of: (1) excision of the damaged base (e.g. 8-oxoguanine), (2) incision of the DNA backbone at the abasic site product, (3) removal of the abasic terminal fragment, (4) gap-filling synthesis, and (5) ligation of the final nick. Our focus has been to understand the molecular mechanisms of BER for two common oxidative DNA damage intermediates, specifically abasic sites and DNA strand breaks that harbor non-conventional 3?-blocking termini (e.g. phosphates). 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 abasic sites, is a structure-specific endonuclease that scans DNA for a unique flexibility associated with the abasic lesion. While this protein operates as the predominant (if not only) mammalian enzyme in abasic site repair, we have shown that it has a more limited role in the excision of 3?-blocking damages, depending on DNA context/structure; thus other proteins likely contribute to this corrective process. Presently, we are determining the mechanism by which Ape1 cuts DNA (the first step in removing the abasic damage) and communicates with other proteins in the BER pathway, most notably DNA polymerase beta and Xrcc1, using biochemical, NMR spectroscopy and crystallography techniques. Our structure-function analysis of proteins in BER is now being expanded into understanding the impact of genetic variation found in the human population on DNA repair function. The hypothesis is that certain genetic differences will produce proteins that are less effective at DNA repair, thus rendering the affected individual more susceptible to environmental or food agent exposures that induce oxidative stress and increase oxidative damage. We have recently shown that indeed genetic differences in APE1 can lead to proteins with reduced repair efficiency. 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。然而，在此过程中，代谢副产品统称为活性氧（也称为自由基）。这些反应性产物攻击包括脂质，蛋白质和DNA在内的各种细胞成分。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。这样的终点与人类疾病，最著名的是癌症和神经退行性相关，以及衰老过程。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，可依次切除目标损伤并将DNA恢复为原始的，未修饰的形式。简而言之，BER的主要步骤包括：（1）切除受损的底座（例如8-氧甲烷），（2）在Abasic位点产物处切开DNA骨干线，（3）去除可覆盖末端片段，（4）GAP填充填充合成，以及（5）最终刻入最终刻盘的连接。我们的重点是了解两个常见的氧化DNA损伤中间体BER的分子机制，特别是无碱性位点和DNA链破裂，这些损伤含有非惯性3？-Blocking Termini（例如磷酸盐）。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BER的中心参与者和Abasic部位的主要哺乳动物修复蛋白，是一种特定于结构的核酸酶，它扫描DNA，以确保DNA具有与阿巴斯病变相关的独特灵活性。尽管该蛋白在无碱性位点修复中作为主要（如果不仅）哺乳动物酶起作用，但我们已经表明，它在切除3？块损伤中的作用更为有限，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。目前，我们正在确定APE1剪切DNA的机制（消除无碱损伤的第一步）并与BER途径中的其他蛋白质进行通信，最著名的是使用生物化学，NMR光谱和晶体学技术，最著名的是DNA聚合酶β和XRCC1。现在，我们对BER中蛋白质的结构功能分析正在扩展到了解人口中发现的遗传变异对DNA修复功能的影响。假设是某些遗传差异会产生在DNA修复方面效率较低的蛋白质，从而使受影响的个体更容易受到环境或食物剂的暴露，从而诱导氧化应激并增加氧化损伤。我们最近表明，APE1的遗传差异确实可以导致蛋白质的修复效率降低。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。