Oxidative DNA Damage And Its Processing

DNA氧化损伤及其处理

• 批准号: 7132318
• 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/7132318
• 关键词: DNA damage; DNA repair; N glycosidase; aging; cell line; cyclin dependent kinase; developmental genetics; dwarfism; enzyme activity; gene mutation; mitochondrial DNA; molecular genetics; molecular pathology; oxidative stress; pentosyltransferase; phosphorylation; posttranslational modifications; protein protein interaction

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 resynthesized, the most significant consequence of 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 are formed after oxidative stress. High levels of these lesions are strongly associated with the development of cancer and 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 the mitochondrial DNA rather than the nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve the base excision (BER) and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarifying these pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes. Using in vitro assays we are able to examine the repair of different types of lesions and to measure different steps of the pathway. Furthermore, we can measure DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. We and others identified several protein interactions for the core BER enzymes. These protein interactions are physical and functional and together support the "passing of baton" model, in which BER 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 finding new protein partners in this process, including poly(ADP) ribose polymerase (PARP), which is activated by DNA strand breaks. We are further examining these interactions in a human disorder, Cockayne syndrome (CS), characterized by premature aging. There are deficiencies in the repair of oxidative DNA damage in the nuclear and mitochondrial DNA of CS cells, and this may be the major underlying cause of the disease. We recently demonstrated that the CSB protein, mutated in CS patients, interacts with PARP1 and that these two proteins cooperate in the cellular responses to oxidative stress. One base lesion, 8-oxoG, is of special interest since it causes mutations, if left unrepaired. We have studied the mechanism of repair of this lesion and find that it is repaired mainly via BER and in a mode that is not coupled to transcription. We also find that the major DNA glycosylase for 8-oxoG repair, oxoguanine DNA glycosylase (OGG1), interacts with and can be phosphorylated by the cyclin-dependent kinase cdk4. This post-translational modification modulates OGG1 catalytic activity, suggesting a role for signaling pathways in the response to oxidative DNA damage.

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被降解和重新合成，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。氧化应激后形成许多不同的DNA碱基变化。这些损伤的高水平与癌症的发展密切相关，并与衰老过程有关。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(BER)和核苷酸切除修复(NER)途径，目前人们对澄清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。使用体外试验，我们能够检查不同类型的损伤的修复，并测量途径的不同步骤。此外，我们还可以在核DNA和线粒体DNA中分别测量DNA损伤过程。我们和其他人确定了核心BER酶的几种蛋白质相互作用。这些蛋白质相互作用是物理和功能的，共同支持“传递接力棒”模型，在该模型中，BER在不同步骤中发生，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。我们正在这个过程中找到新的蛋白质伙伴，包括多聚(ADP)核糖聚合酶(PARP)，它是由DNA链断裂激活的。我们正在进一步研究以过早衰老为特征的人类疾病--Cockayne综合征(CS)中的这些相互作用。CS细胞的核和线粒体DNA在氧化损伤修复方面存在缺陷，这可能是该病的主要原因。我们最近证明，在CS患者中突变的CSB蛋白与PARP1相互作用，这两种蛋白在细胞对氧化应激的反应中相互作用。其中一个碱基损伤8-oxoG特别令人感兴趣，因为如果不修复，它会导致突变。我们研究了这种损伤的修复机制，发现它主要通过BER修复，并且以一种与转录无关的方式修复。我们还发现，8-oxoG修复的主要DNA糖基酶，氧鸟嘌呤DNA糖基酶(OGG1)，与细胞周期蛋白依赖的激酶CDK4相互作用，并可被其磷酸化。这种翻译后修饰调节了OGG1的催化活性，表明信号通路在对DNA氧化损伤的反应中发挥了作用。