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，在这个过程中，代谢副产物形成，统称为活性氧物种(也称为自由基)。这些反应产物攻击各种细胞成分，包括脂质、蛋白质和DNA。与DNA的反应，也就是我们的遗传物质，可能会导致几种损害中间体。如果不加以修复，这种损伤可能会促进不必要的基因变化或导致细胞死亡。这些终点与人类疾病有关，最明显的是癌症和神经退化，以及衰老过程。为了调节这些结果，生物体进化出了一系列修复系统，可以识别和移除特定形式的DNA损伤。碱基切除修复(BER)是修复DNA氧化损伤的主要途径，涉及多个蛋白质的协同作用，这些蛋白质依次切除靶损伤并将DNA恢复到其原始的未经修饰的形式。简而言之，BER的主要步骤包括：(1)切除受损的碱基(如8-氧鸟嘌呤)，(2)在基本位点产物处切断DNA骨架，(3)去除基本末端片段，(4)缝隙填充合成，(5)连接最终缺口。我们的重点是了解两种常见的DNA氧化损伤中间产物的BER的分子机制，特别是含有非常规3？封闭末端(如磷酸盐)的碱性位点和DNA链断裂。为此，我们分离了几个BER蛋白参与者，并正在定义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，也是哺乳动物基本部位的主要修复蛋白，是一种结构特异性内切酶，它扫描DNA以寻找与基本损伤相关的独特灵活性。虽然该蛋白在基本部位修复中作为哺乳动物的主要酶起作用(如果不是唯一的话)，但我们已经证明，它在切除3？-阻断损伤方面的作用更加有限，这取决于DNA上下文/结构；因此，其他蛋白质可能有助于这一纠正过程。目前，我们正在利用生化、核磁共振和结晶学技术来确定APE1切割DNA(消除基本损伤的第一步)以及与BER途径中的其他蛋白质，尤其是DNA聚合酶β和Xrcc1进行通信的机制。我们对BER中蛋白质的结构-功能分析现在正在扩展，以了解在人类群体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DNA修复效率较低的蛋白质，从而使受影响的个体更容易受到环境或食品剂暴露的影响，这些环境或食品剂会导致氧化应激并增加氧化损伤。我们最近已经证明，APE1中的遗传差异确实会导致修复效率降低的蛋白质。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损伤反应系统中的基因变异与人类疾病和衰老过程的关系。