Mitochondrial DNA Repair Processes In Oxidative Stress And Aging

氧化应激和衰老中的线粒体 DNA 修复过程

• 批准号: 7964030
• 负责人: Vilhelm Bohr
• 金额: $ 71.81万
• 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/7964030
• 关键词: 8-hydroxyguanosine; Affinity; Affinity Chromatography; Age; Aging; Base Excision Repairs; Binding; Biochemical; Biological Process; Cell Death; Cell Nucleus; Cells; Complex; DNA; DNA Binding; DNA Repair; DNA Repair Pathway; DNA glycosylase; DNA lesion; Deletion Mutation; Electron Transport; Enzymes; Excision; Genes; Genetic Recombination; Homologous Gene; Human; In Vitro; Investigation; Lead; Lesion; Mammalian Cell; Mass Spectrum Analysis; Metabolism; Mismatch Repair; Mitochondria; Mitochondrial DNA; OGG1 gene; Organelles; Oxidative Stress; Pathway interactions; Peptides; Play; Process; Protein Isoforms; Proteins; RNA Interference; Reactive Oxygen Species; Recombinants; Regulation; Resistance; Role; Set protein; Specificity; Structure; base; cytochrome c oxidase; endonuclease III; enzyme activity; helicase; in vitro Assay; mitochondrial genome; mutant; oxidative DNA damage; oxidative damage; repair enzyme; repaired; telomere; transcription factor

The base excision repair pathway is initiated by the action of a class of enzymes known as DNA glycosylases, which recognize and release the damaged base, and thus give specificity to the repair process. Mammalian cells carry two major DNA glycosylases for the repair of oxidized bases, oxoguanine DNA glycosylase (OGG1) and Endonuclease III homologue (NTH1). We found that OGG1 plays a crucial role in the repair of oxidized lesions in mitochondria and is probably the only DNA glycosylase for 8-oxoG removal in these organelles. In human cells two distinct OGG1 isoforms are expressed, alpha and beta. Because of the high abundance of the b-OGG1 protein in human mitochondria we are now investigating whether it has any biological function. All BER enzymes are encoded in the nucleus and transported to mitochondria; however there is very limited information on the regulation of mitochondrial BER. In mammalian mitochondria the mtDNA is found in a large protein-DNA complex known as the nucleoid. One of the most abundant protein components of mammalian nucleoids is the transcription factor TFAM, which has been postulated to have a structural function in compacting the mtDNA into the nucleoid structure. Using recombinant human TFAM we are now investigating whether TFAM modulates mtDNA repair. We find that that TFAM may modulate BER proteins through an as yet undetermined mechanism. To explore whether it is a function of TFAMs high affinity for DNA we have created TFAM DNA binding mutants and are re-evaluating the activity of the BER enzyme activities in the presence of this mutant. Additionally, we are exploring whether TFAM physically interacts with any mitochondrial BER proteins. We are now investigating whether mammalian mitochondria have any of the other repair pathways that operate in the nucleus, such as mismatch repair (MMR). Our results show that human mitochondria can catalyze mismatch repair in vitro and contain a mismatch binding activity. Using affinity purification with a mismatch-containing DNA substrate, and mass spectrometry-peptide analyses we identified 3 proteins in the mismatch-bound complex, the transcription factor YB-1, the Cytochrome oxidase-assembly factor LRP130 and an UV-resistance associated gene of unknown activity. We showed mitochondrial localization of YB-1 using both the endogenous as well as ectopic expressed protein. Interestingly, abrogation of YB1 levels by RNA interference significantly decreased mitochondrial-catalysed mismatch repair activity in an in vitro assay, indicating that this protein is involved in mitochondiral MMR. These observations, along with results from others clearly establish that mammalian mitochondria have a functional mismatch repair pathway. Another important set of proteins involved in mitochondrial DNA metabolism are the helicases SUV3 and PIF1. We have investigated the biochemical functions of SUV3, and it appears to interact with some mitochondrial and telomere proteins, making it possible that it functions both in telomeres and in mitochondria. This is under further investigation. While Oxidative damage processing is very efficient in mitochondria, little is known about the recombination DNA repair pathways in these organelles. Interestingly, we detect direct functional interactions between the OGG1 protein, present in the nucleus and in mitochondria, and the recombination protein RAD52. It is not known whether RAD52 is present in mitochondria, and this is currently under investigation.

碱基切除修复途径是由一类称为DNA糖基化酶的酶的作用启动的，所述酶识别并释放受损的碱基，从而为修复过程提供特异性。哺乳动物细胞携带用于修复氧化碱基的两种主要DNA糖基化酶，氧代鸟嘌呤DNA糖基化酶（OGG 1）和内切核酸酶III同源物（NTH 1）。我们发现OGG 1在线粒体氧化损伤的修复中起着至关重要的作用，并且可能是这些细胞器中唯一用于去除8-oxoG的DNA糖基化酶。在人类细胞中，表达两种不同的OGG 1亚型，α和β。由于b-OGG 1蛋白在人类线粒体中的高丰度，我们现在正在研究它是否具有任何生物学功能。 所有BER酶都在细胞核中编码并转运到线粒体;然而，关于线粒体BER调控的信息非常有限。在哺乳动物的线粒体中，线粒体DNA存在于一个大的蛋白质-DNA复合物中，称为类核。哺乳动物类核中最丰富的蛋白质组分之一是转录因子TFAM，它被认为具有将mtDNA压缩成类核结构的结构功能。使用重组人TFAM，我们现在正在研究TFAM是否调节mtDNA修复。我们发现TFAM可能通过一种尚未确定的机制调节BER蛋白。为了探索TFAM对DNA的高亲和力是否是一种功能，我们已经创建了TFAM DNA结合突变体，并正在重新评估BER酶活性在该突变体存在下的活性。此外，我们正在探索TFAM是否与任何线粒体BER蛋白发生物理相互作用。 我们现在正在研究哺乳动物线粒体是否有任何其他在细胞核中运作的修复途径，如错配修复（MMR）。我们的研究结果表明，人线粒体可以催化体外错配修复，并含有错配结合活性。使用含有错配的DNA底物的亲和纯化和质谱肽分析，我们确定了3个蛋白质的错配结合的复合物，转录因子YB-1，细胞色素氧化酶组装因子LRP 130和未知活性的UV抗性相关基因。我们显示了线粒体定位的YB-1使用的内源性以及异位表达的蛋白质。有趣的是，通过RNA干扰消除YB 1水平显著降低了体外试验中的细胞催化的错配修复活性，表明该蛋白质参与线粒体MMR。这些观察结果，沿着来自其他人的结果清楚地建立了哺乳动物线粒体具有功能性错配修复途径。 参与线粒体DNA代谢的另一组重要蛋白质是解旋酶SUV 3和PIF 1。我们已经研究了SUV 3的生化功能，它似乎与一些线粒体和端粒蛋白相互作用，使其有可能在端粒和线粒体中发挥作用。此事正在进一步调查中。 虽然氧化损伤处理在线粒体中非常有效，但对这些细胞器中的重组DNA修复途径知之甚少。有趣的是，我们检测到OGG 1蛋白，存在于细胞核和线粒体，和重组蛋白RAD 52之间的直接功能相互作用。目前尚不清楚RAD 52是否存在于线粒体中，目前正在研究中。