Repair Mechanisms For Strand Breaks in DNA

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质（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（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。这些终点与人类疾病有关，最明显的是癌症和神经变性，以及衰老过程。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。简言之，BER的主要步骤包括：（1）切除受损碱基（例如8-氧代鸟嘌呤），（2）在脱碱基位点产物处切割DNA骨架，（3）去除脱碱基末端片段，（4）缺口填充合成，和（5）连接最终切口。我们的重点一直是了解BER的分子机制，为两个常见的氧化DNA损伤中间体，特别是脱碱基位点和DNA链断裂，港口非传统的3？封闭末端（例如磷酸盐）。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，也是哺乳动物脱碱基位点的主要修复蛋白，它是一种结构特异性核酸内切酶，可以扫描DNA，寻找与脱碱基损伤相关的独特灵活性。虽然这种蛋白质作为主要的（如果不是唯一的）哺乳动物酶在脱碱基位点修复，我们已经表明，它有一个更有限的作用，在切除3？阻断损伤，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。目前，我们正在确定Ape 1切割DNA（去除脱碱基损伤的第一步）并与BER途径中的其他蛋白质（最值得注意的是DNA聚合酶β和Xrcc 1）进行通信的机制，使用生物化学，NMR光谱学和晶体学技术。我们对BER中蛋白质的结构-功能分析现在正在扩展到理解人类群体中发现的遗传变异对DNA修复功能的影响。该假说认为，某些遗传差异会产生在DNA修复方面不太有效的蛋白质，从而使受影响的个体更容易受到环境或食物因子暴露的影响，从而诱导氧化应激并增加氧化损伤。我们最近发现，APE 1的遗传差异确实会导致蛋白质的修复效率降低。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。

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.