Oxidative Dna Damage And Its Processing

工作总结：生物体不断地暴露在来自环境因素和内源性代谢过程的氧化应激下。由此产生的氧化修饰发生在蛋白质、脂类和DNA中。由于蛋白质和脂类很容易降解和合成，氧化应激最重要的后果被认为是DNA修饰，这种修饰可以通过突变和其他类型的基因组不稳定的形成而成为永久性的。在某种形式的氧化应激之后，已经看到了许多不同的DNA碱基变化，这些损伤被广泛认为是癌症发展的诱因，也与衰老过程有关。几项研究证明，氧化DNA损伤随着年龄的增长而积累，似乎这种积累的主要部位是线粒体DNA，而不是核DNA。与清除氧化DNA损伤有关的DNA修复机制比以前认为的要复杂得多。它们涉及碱基切除修复(BER)途径和核苷酸切除修复(NER)途径，目前人们对阐明这些途径及其相互作用非常感兴趣。我们使用了许多不同的方法来探索修复过程的机制，我们能够检查不同类型的损伤的修复，并测量修复过程的不同步骤。此外，我们还可以测量DNA在核DNA和线粒体DNA中的损伤过程。我们已经建立了使用细胞核提取物的体外检测方法，以测量DNA氧化损伤的碱基切除修复过程。实验表明，碱基切除修复有两种形式，根据DNA中新合成碱基的长度来区分。我们已经证明DNA聚合酶β在这两个过程中都是被分解的，并且它发挥着关键作用，可能是在与FLAW核酸内切酶FEN-1的相互作用中。我们已经揭示了碱基切除修复途径中涉及的蛋白质之间的其他一些蛋白质相互作用。我们已经在体外重建了这一过程，并正在探索物理和功能蛋白质的相互作用。这表明存在一个主要的碱基切除修复复合体，包括AP内切酶、增殖细胞核抗原、聚合酶(S)、糖基酶(S)等。这些蛋白质相互作用是物理和功能的，共同支持“传递Buton”模型，在该模型中，碱基切除修复在不同步骤中进行，由单个蛋白质相互作用支持，这些蛋白质相互作用是修复复合体的组成部分，可能位于DNA损伤处。我们正在进一步研究这些相互作用在以过早衰老为特征的人类疾病Cockayne综合征(CS)中，核DNA中氧化DNA损伤的修复似乎存在缺陷，这可能是该疾病的主要潜在原因。

Summary of work: Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resyn-thesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as in- stigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is cur-rently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, and we are able to examine the repair of different types of lesions and to meas-ure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. We have established in vitro assays using nuclear extracts from cells to measure the process of base excision repair of oxidative DNA damage. The experiments show that there are two forms of Base excision repair distinguishable by the length of the newly synthesized bases in DNA. We have shown that DNA polymerase beta is in volved in both of these processes, and that it plays a critical role, possibly in interaction with FLAP endonuclease FEN-1. We have disclosed a number of other protein interaction between proteins involved in the Base excision repair pathway. We have reconstituted this process in vitro and are exploring both physical and functional protein interactions. This suggests that a major base excision repair complex exists which includes AP endonuclease, proliferating cell nuclear antigen (PCNA), polymerase (s), glycosylase(s) and others. These protein interactions are physical and functional and together support the "passing of buton" model in which base excision repair takes place in different steps supported by individual protein interactions that are components of a repair complex, possibly situated at the DNA lesion. We are further examining these inetractionsIn a human disorder, Cockayne syndrome (CS), characterized by premature aging, there appear to be deficiencies in the repair of oxidative DNA damage in the nuclear DNA, and this may be the major underlying cause of the disease.