OXIDATIVE DNA DAMAGE AND ITS PROCESSING

DNA氧化损伤及其处理

• 批准号: 6431453
• 负责人: Vilhelm A Bohr
• 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/6431453
• 关键词: DNA damage; DNA repair; cell line; chromatography; deoxyguanosine; free radical oxygen; mitochondrial DNA; oxidative stress; photoactivation; radiation genetics; ultraviolet radiation; xeroderma pigmentosum

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.

工作总结：生物体不断暴露于环境药物和内源代谢过程中的氧化应激。所得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融合，因此氧化应激的最重要后果被认为是DNA的修饰，可以通过突变和其他类型的基因组不稳定性形成来永久性。 遵循某种形式的氧化应激，已经看到了许多不同的DNA碱基变化，并且这些病变被广泛认为是癌症发展的刺激器，也与衰老过程有关。 几项研究表明，氧化性DNA病变随着衰老而累积，看来该积累的主要部位是线粒体DNA而不是核DNA。 去除氧化DNA病变的DNA修复机制比以前考虑的要复杂得多。 它们涉及基础切除修复（BER）途径和核苷酸切除修复（NER）途径，目前，人们对阐明途径及其相互作用有很大的兴趣。 我们已经使用了多种不同的方法来探索修复过程的机制，并且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，我们可以测量核DNA中的DNA损伤处理，并在线粒体DNA中分别测量。 我们已经使用细胞中的核提取物建立了体外测定，以测量氧化性DNA损伤的碱基切除修复过程。实验表明，有两种形式的碱性切除修复可以通过DNA中新合成碱的长度来区分。我们已经表明，在这两个过程中，DNA聚合酶β都在体积中，并且它在与皮瓣核酸内切酶Fen-1的相互作用中起着至关重要的作用。我们已经披露了与基础切除修复途径有关的蛋白质之间的许多其他蛋白质相互作用。 我们已经在体外重构了这一过程，并正在探索物理和功能蛋白质相互作用。 这表明存在主要的碱基切除修复复合物，其中包括AP内切核酸酶，增殖细胞核抗原（PCNA），聚合酶，聚合酶，糖基酶等。我们还建立了一种用于测量线粒体DNA修复的体外测定法。在这里，我们还表征了DNA修复贴片的大小，并发现在线路上只有短长度贴片大小类型的修复。与广泛持有的概念相反，线粒体对氧化DNA损伤具有有效的DNA修复，我们正在探索机制。 在人类疾病中，Cockayne综合征（CS）以过早的衰老为特征，在核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根本原因。