OXIDATIVE DNA DAMAGE AND ITS PROCESSING

工作概述：生物体不断暴露于环境因素和内源性代谢过程的氧化应激。由此产生的氧化修饰发生在蛋白质、脂质和DNA中。由于蛋白质和脂质很容易降解和重新合成，氧化应激最重要的后果被认为是DNA修饰，它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，已经发现了许多不同的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。一些研究已经证明，氧化性DNA损伤随着年龄的增长而积累，而且这种积累的主要部位似乎是线粒体DNA，而不是核DNA。DNA修复机制涉及DNA氧化损伤的清除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，目前对这些途径及其相互作用的澄清有很大的兴趣。我们已经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病变的修复并测量修复过程的不同步骤。此外，我们还可以测量核DNA和线粒体DNA中的DNA损伤过程。我们已经建立了体外实验，使用细胞核提取物来测量碱基切除修复氧化DNA损伤的过程。实验表明，根据DNA中新合成碱基的长度可以区分碱基切除修复的两种形式。我们已经证明DNA聚合酶β参与了这两个过程，并且它起着关键作用，可能与FLAP内切酶FEN-1相互作用。我们已经揭示了一些其他蛋白质之间的相互作用，涉及碱基切除修复途径。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（PCNA）、聚合酶(s)、糖基化酶(s)等。我们还建立了一种体外测定线粒体DNA修复的方法。在这里，我们还表征了DNA修复补丁大小，并发现在线粒体中只有短长度补丁大小类型的修复。与广泛持有的观念相反，线粒体对氧化DNA损伤具有有效的DNA修复作用，我们正在探索其机制。在人类疾病中，柯凯因综合征（CS），以过早衰老为特征，在细胞核DNA的氧化DNA损伤修复中似乎存在缺陷，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 instigators 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 currently 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 measure 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. We have also established an in vitro assay for measuring DNA repair in mitochondria. Here we also characterize the DNA repair patch size and find that in the mitochodria there is only repair of the short length patch size type. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage and we are exploring the mechanisms. In 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.