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。由于蛋白质和脂质容易降解和再合成，氧化应激的最重要后果被认为是DNA修饰，这可以通过形成突变和其他类型的基因组不稳定性而成为永久性的。氧化应激后形成许多不同的DNA碱基变化。高水平的这些病变与癌症的发展密切相关，并与衰老过程有关。一些研究已经证明，氧化DNA损伤随着衰老而积累，并且似乎这种积累的主要部位是线粒体DNA而不是核DNA。DNA修复机制涉及去除氧化DNA损伤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，目前有很大的兴趣在澄清这些途径及其相互作用。我们已经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病变的修复，并测量途径的不同步骤。此外，我们可以测量核DNA中的DNA损伤过程，并分别测量线粒体DNA中的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的和功能性的，并且共同支持“接力棒传递”模型，其中BER在不同的步骤中发生，这些步骤由作为修复复合物的组分的个体蛋白质相互作用支持，可能位于DNA损伤处。我们正在发现这个过程中的新蛋白质伙伴，包括聚（ADP）核糖聚合酶（PARP），它被DNA链断裂激活。我们正在进一步研究这些相互作用在人类疾病，Cockayne综合征（CS），其特征是过早衰老。CS细胞的核和线粒体DNA中存在氧化DNA损伤修复的缺陷，这可能是疾病的主要潜在原因。我们最近证明了CS患者中突变的CSB蛋白与PARP 1相互作用，并且这两种蛋白在细胞对氧化应激的反应中合作。一个基础病变，8-oxoG，是特别感兴趣的，因为它会导致突变，如果不修复。我们研究了这种损伤的修复机制，发现它主要通过BER修复，并且以不与转录偶联的模式修复。我们还发现，8-oxoG修复的主要DNA糖基化酶，氧鸟嘌呤DNA糖基化酶（OGG 1），与细胞周期蛋白依赖性激酶cdk 4相互作用，并可被其磷酸化。这种翻译后修饰调节OGG 1的催化活性，表明信号通路在氧化DNA损伤反应中的作用。