Mitochondrial DNA Repair Processes In Oxidative Stress And Aging

碱基切除修复途径是由一类称为DNA糖基酶的酶启动的，它识别并释放受损的碱基，从而赋予修复过程特异性。哺乳动物细胞携带两种主要的DNA糖基酶，用于修复氧化碱基，氧鸟嘌呤DNA糖基酶(OGG1)和核酸内切酶III同源酶(NTH1)。我们发现OGG1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能清除8-oxoG的DNA糖基酶。在人类细胞中，表达了两种不同的OGG1亚型，即α和β。由于b-OGG1蛋白在人类线粒体中的丰度很高，我们现在正在研究它是否具有生物学功能。 所有的BER酶都在细胞核中编码，并运输到线粒体；然而，关于线粒体BER的调控信息非常有限。在哺乳动物的线粒体中，线粒体DNA存在于一个被称为类核的大型蛋白质-DNA复合体中。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。利用重组人TFAM，我们现在正在研究TFAM是否调节线粒体DNA修复。我们发现，TFAM可能通过一种尚未确定的机制来调节BER蛋白。为了探索这是否是TFAM与DNA高亲和力的函数，我们创建了TFAM DNA结合突变体，并正在重新评估该突变体存在时BER酶的活性。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工作的修复途径，如错配修复(MMR)。我们的结果表明，人线粒体在体外可以催化错配修复，并包含错配结合活性。利用含有错配的DNA底物亲和纯化和质谱肽分析，我们在错配结合的复合体中鉴定了3个蛋白质，转录因子YB-1，细胞色素氧化酶组装因子LRP130和一个活性未知的抗紫外线相关基因。我们使用内源和异源表达的蛋白显示了YB-1的线粒体定位。有趣的是，在体外实验中，通过RNA干扰降低YB1水平显著降低了线粒体催化的错配修复活性，表明该蛋白参与了线粒体MMR。这些观察结果以及其他研究结果清楚地表明，哺乳动物线粒体存在功能错配修复途径。 参与线粒体DNA新陈代谢的另一组重要蛋白质是解旋酶SUV3和PIF1。我们已经研究了SUV3的生化功能，它似乎与一些线粒体和端粒蛋白相互作用，使其有可能同时在端粒和线粒体中发挥作用。这件事正在进一步调查中。 虽然线粒体的氧化损伤处理是非常有效的，但对这些细胞器中的重组DNA修复途径知之甚少。有趣的是，我们检测到存在于细胞核和线粒体中的OGG1蛋白与重组蛋白RAD52之间的直接功能相互作用。目前尚不清楚线粒体中是否存在RAD52，目前正在进行调查。

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.