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: 7592045
• 负责人: Vilhelm A Bohr
• 金额: $ 97.32万
• 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/7592045
• 关键词: 8-hydroxyguanine; 8-hydroxyguanosine; Accounting; Affect; Affinity; Affinity Chromatography; Age; Aging; Amino Acids; Base Excision Repairs; Binding; Biological; Biological Assay; Biological Process; Cell Death; Cell Nucleus; Cells; Class; Complex; DNA; DNA Binding; DNA Repair; DNA glycosylase; DNA lesion; Deletion Mutation; Electron Transport; Electrostatics; End Point; Enzymes; Excision; Genes; Homologous Gene; Human; In Vitro; Lead; Lesion; Liver; Localized; Mammalian Cell; Mass Spectrum Analysis; Measures; Membrane; Mismatch Repair; Mitochondria; Mitochondrial DNA; Mus; Nature; Nuclear; OGG1 gene; Organelles; Oxidases; Oxidative Stress; Pathway interactions; Peptides; Play; Post-Translational Protein Processing; Process; Protein Isoforms; Proteins; Pyrimidine; Pyrimidines; RNA Interference; Reactive Oxygen Species; Recombinants; Regulation; Repair Complex; Resistance; Role; Site-Directed Mutagenesis; Sodium Chloride; Specificity; Techniques; aqueous; base; endonuclease III; established cell line; in vitro Assay; interest; mitochondrial genome; mouse model; oxidative DNA damage; repair enzyme; repaired; rho; thymine glycol; 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. 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.

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，这些酶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 糖基化酶，用于修复氧化碱基，氧化鸟嘌呤 DNA 糖基化酶 (OGG1) 和核酸内切酶 III 同源物 (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去除 8-oxoG 的 DNA 糖基化酶。我们还表明，NTH 定位于线粒体，参与去除氧化嘧啶。我们研究的一大亮点是我们利用色谱技术检测 DNA 修复活性并测量 DNA 损伤的实际发生情况。我们分析了小鼠肝脏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。使用缺乏这些糖基化酶的小鼠模型，我们发现 8-oxoG 和 Fapy-G 在 OGG1-/- 小鼠的 DNA 中积累，FapyG 和 fapyadenine (FapyA) 在 NTH1-/- 小鼠的 DNA 中积累。我们还表明，FapyG 和 FapyA 是由同一组 DNA 糖基化酶修复的，这些酶可以在细胞核和线粒体中从 DNA 中去除 8-oxoG 和胸腺嘧啶二醇。这些结果表明，这些病变的积累可能会产生重要的生物学后果，至少与 8-oxoG 的后果一样相关。此外，我们还建立了新鉴定的DNA糖基化酶NEIL1的线粒体定位，该酶对开环底物具有更高的特异性。 在人类细胞中表达两种不同的 OGG1 亚型：α 和 β。 Beta-OGG1 仅定位于线粒体，并被认为提供 8-oxoG 糖基化酶活性。我们纯化了重组b-OGG1，发现该蛋白缺乏糖基化酶活性。定点诱变研究发现，b-同工型中存在两种氨基酸，它们使 a-同工型失活。我们还发现大约 10% 的 a-OGG1 定位于线粒体，并可能解释线粒体 8-oxoG 糖基化酶活性。由于人类线粒体中 b-OGG1 蛋白的丰度很高，我们现在正在研究它是否具有任何生物学功能。为此，我们正在建立具有 b-OGG1 同种型特异性稳定敲低的细胞系，以便确定在缺乏该蛋白质的情况下可能发生的生物学终点变化。 所有 BER 酶均在细胞核中编码并转运至线粒体；然而，关于线粒体 BER 调节的信息非常有限。我们测量了缺乏 mtDNA (rho-) 的线粒体中的 BER 活性。尽管不存在线粒体DNA，但线粒体中存在一套完整的BER酶，并且与野生型线粒体相比，大多数活性仅略有下降。有趣的是，核 BER 活动也受到 mtDNA 缺失的影响，这表明两个区室中的 BER 之间存在有趣的串扰。线粒体由两层膜（外膜和内膜）组成，包围着水性基质室。我们研究了线粒体中 BER 的空间组织，发现大多数 BER 活性不能自由溶解在基质中，而是与膜部分相关。这种关联本质上可能是静电的，因为它会被高盐浓度破坏。这种高阶 DNA 修复复合物的存在对 mtDNA 修复具有深远的影响，因为它暗示了 DNA 流经这种固定复合物的机制。在哺乳动物线粒体中，mtDNA 存在于称为类核的大型蛋白质-DNA 复合物中。哺乳动物核仁中最丰富的蛋白质成分之一是转录因子 TFAM，人们认为它具有压缩核仁中 mtDNA 的结构功能。我们现在正在使用重组人 TFAM 研究 TFAM 是否调节 mtDNA 修复。我们发现 TFAM 以更高的亲和力与含有氧化碱基的 DNA 结合，并且当 TFAM 结合时，BER 酶的催化活性降低，很可能是因为对受损碱基的可及性较差。这些结果表明 TFAM 可能通过翻译后修饰改变其 DNA 结合亲和力来调节 BER。 我们现在正在研究哺乳动物线粒体是否具有在细胞核中起作用的任何其他修复途径，例如错配修复（MMR）。我们的研究结果表明，人类线粒体可以在体外催化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，我们鉴定了错配结合复合物中的 3 种蛋白质：转录因子 YB-1、细胞色素氧化酶组装因子 LRP130 和 UV 抗性相关基因未知的活动。我们使用内源性和异位表达的蛋白质展示了 YB-1 的线粒体定位。有趣的是，在体外试验中，通过 RNA 干扰消除 YB1 水平显着降低了线粒体催化的错配修复活性，表明该蛋白参与线粒体 MMR。这些观察结果以及其他结果清楚地表明哺乳动物线粒体具有功能性错配修复途径。