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.

工作摘要：生物体不断暴露于来自环境因素和内源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，氧化应激最显着的后果被认为是 DNA 修饰，这种修饰可以通过突变的形成和其他类型的基因组不稳定性而变得永久性。 在某种形式的氧化应激后，我们发现了许多不同的 DNA 碱基变化，这些病变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 损伤会随着衰老而积累，而且这种积累的主要部位似乎是线粒体 DNA，而不是核 DNA。 去除氧化性 DNA 损伤所涉及的 DNA 修复机制比之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。 我们使用了多种不同的方法来探索修复过程的机制，并且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，我们可以分别测量核 DNA 和线粒体 DNA 中的 DNA 损伤处理。 我们已经建立了使用细胞核提取物的体外测定法来测量氧化 DNA 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，可根据 DNA 中新合成碱基的长度进行区分。我们已经证明 DNA 聚合酶 β 参与了这两个过程，并且发挥着关键作用，可能与 FLAP 核酸内切酶 FEN-1 相互作用。我们已经公开了参与碱基切除修复途径的蛋白质之间的许多其他蛋白质相互作用。 我们已经在体外重建了这一过程，并正在探索物理和功能性蛋白质相互作用。 这表明存在主要的碱基切除修复复合物，其中包括AP核酸内切酶、增殖细胞核抗原(PCNA)、聚合酶、糖基化酶等。我们还建立了一种测量线粒体 DNA 修复的体外测定方法。在这里，我们还表征了 DNA 修复补丁的大小，发现在线粒体中仅存在短长度补丁大小类型的修复。与广泛持有的观念相反，线粒体能够有效修复氧化性 DNA 损伤，我们正在探索其机制。 在以过早衰老为特征的人类疾病科凯恩综合征 (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根本原因。