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。我们还表明，nth定位于线粒体，在那里它参与清除氧化的嘧啶。我们研究的一个优点是我们检测DNA修复活动，并使用层析技术测量DNA损伤的实际发生情况。我们分析了小鼠肝DNA中8-oxoG和其他氧化碱基的水平，发现开环氧化损伤fapyguanine(FapyG)的水平高于8-oxoG。利用这些糖基酶缺乏的小鼠模型，我们发现8-oxoG和Fapy-G在OGG1-/-小鼠的DNA中积累，而FapyG和FapyAdine(FapyA)在NTH1-/-小鼠的DNA中积累。我们还表明，FapyG和FapyA是由同一组DNA糖基酶修复的，这些糖基酶从DNA中移除8-oxoG和胸腺嘧啶二醇，无论是在细胞核还是在线粒体中。这些结果表明，这些损伤的积累可能具有重要的生物学后果，至少与8-oxoG的相关性相同。此外，我们还建立了新发现的DNA糖基酶NEIL1的线粒体定位，该酶对开环底物具有较高的特异性。 在人类细胞中，表达了两种不同的OGG1亚型，即α和β。β-OGG1仅定位于线粒体，并被认为提供8-oxoG糖基化酶活性。我们对重组b-OGG1进行了纯化，发现该蛋白缺乏糖基酶活性。定点突变研究发现，在b-异构体中发现的两种氨基酸使a-异构体失去活性。我们还发现，大约10%的a-OGG1定位于线粒体，这可能是线粒体8-oxoG糖基酶活性的原因。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。为此，我们正在建立具有b-OGG1同工型特异性稳定敲除的细胞系，以确定在没有这种蛋白的情况下可能发生变化的生物终点。 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。我们测量了缺乏线粒体DNA(Rho-)的线粒体的BER活性。尽管线粒体中没有mtDNA，但线粒体中存在一整套BER酶，与野生型线粒体相比，大多数酶的活性仅略有下降。有趣的是，核误码率活动也受到线粒体DNA缺失的影响，这表明两个隔室的误码率之间存在有趣的串扰。线粒体由两层膜(外层和内层)组成，包围着一个含水的基质隔室。我们研究了线粒体中BER的空间组织，发现大多数BER活性不是自由溶解在基质中的，而是与膜部分有关。这种联系在本质上很可能是静电，因为它可以被高盐浓度破坏。这种高阶DNA修复复合体的存在对线粒体DNA修复有着深远的影响，因为它表明了DNA通过这种固定复合体流动的机制。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，它被认为具有压缩类核中mtDNA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与含有氧化碱基的DNA结合有更高的亲和力，当TFAM结合时，BER酶的催化活性降低，最可能是因为很难接触到受损的碱基。这些结果表明，TFAM可能通过改变其DNA结合亲和力的翻译后修饰来调节BER。 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DNA底物的亲和纯化和质谱肽分析，我们在错配结合复合体中鉴定了3个蛋白质，转录因子YB-1，柑橘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。

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. We have also shown that NTH localizes to mitochondria, where it is involved in removing oxidized pyrimidines. One strong point of our studies is that we assay for DNA repair activities and measure the actual occurrence of the lesions in DNA using chromatographic techniques. We analyzed the levels of 8-oxoG and other oxidized bases in mouse liver DNA and found that the levels of the ring-opened oxidative lesion fapyguanine (FapyG) is higher than that of 8-oxoG. Using mouse models deficient for these glycosylases we find that 8-oxoG and Fapy-G accumulate in DNA from OGG1-/- mouse and that FapyG and fapyadenine (FapyA) accumulate in DNA from NTH1-/- mice. We also show that FapyG and FapyA are repaired by the same set of DNA glycosylases that remove 8-oxoG and thymine glycols from DNA, both in the nucleus and in mitochondria. These results indicate that the accumulation of these lesions may have important biological consequences, at least as relevant as those of 8-oxoG. Moreover, we established the mitochondrial localization of the newly identified DNA glycosylase NEIL1, which has higher specificity for the ring-opened substrates. In human cells two distinct OGG1 isoforms are expressed, alpha and beta. Beta-OGG1 localizes exclusively to mitochondria and was believed to provide the 8-oxoG glcycosylase activity. We purified recombinant b-OGG1 and found that the protein lacks glycosylase activity. Site-directed mutagenesis studies identified two aminoacids that are found in the b-isoform that render the a-isoform inactive. We also found that approximately 10% of a-OGG1 localizes to mitochondria and may account for the mitochondrial 8-oxoG glycosylase activity. Because of the high abundance of the b-OGG1 protein in human mitochondria we are now investigating whether it has any biological function. For this we are establishing cell lines with isoform-specific stable knockdown of b-OGG1, in order to identify possible biological endpoints altered in the absence of this protein. All BER enzymes are encoded in the nucleus and transported to mitochondria; however there is very limited information on the regulation of mitochondrial BER. We measured BER activities in mitochondria that lack mtDNA (rho-). Despite the absence of mtDNA, a complete set of BER enzymes was present in mitochondria, and most activities were only slightly decreased compared to wild type mitochondria. Interestingly, nuclear BER activities were also affected by the absence of mtDNA, suggesting an interesting cross-talk between BER in both compartments. Mitochondria are comprised of two membranes (outer and inner) enclosing an aqueous matrix compartment. We studied the spatial organization of BER in mitochondria and find that most BER activities are not freely soluble in the matrix, but rather associated with the membrane fraction. This association is likely electrostatic in nature, as it can be disrupted by high salt concentration. The existence of this higher order DNA repair complex has profound implications for mtDNA repair, as it suggests a mechanism in which the DNA flows through this stationary complex. 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 in the nucleoid. Using recombinant human TFAM we are now investigating whether TFAM modulates mtDNA repair. We find that TFAM binds with higher affinity to DNA containing oxidized bases, and that when TFAM is bound the catalytic activity of BER enzymes is decreased, most likely because of poor accessibility to the damaged base. These results indicate that TFAM may function to modulate BER through post-translational modifications that change its DNA binding affinity. 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 Citochrome 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.