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: 7732302
• 负责人: Vilhelm A Bohr
• 金额: $ 61.36万
• 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/7732302
• 关键词: 8-hydroxyguanine; 8-hydroxyguanosine; Accounting; Affect; Affinity; Affinity Chromatography; Age; Aging; Amino Acids; Base Excision Repairs; Binding; Biochemical; 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; Investigation; Lead; Lesion; Liver; Localized; Mammalian Cell; Mass Spectrum Analysis; Measures; Membrane; Metabolism; 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; Set protein; Site-Directed Mutagenesis; Sodium Chloride; Specificity; Techniques; aqueous; base; endonuclease III; established cell line; helicase; in vitro Assay; interest; mitochondrial genome; mouse model; oxidative DNA damage; repair enzyme; repaired; rho; telomere; 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. 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.

碱切除修复途径是由称为DNA糖基化酶的一类酶的作用引发的，该酶识别和释放受损的碱，从而对修复过程具有特殊性。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，氧气DNA DNA糖基化酶（OGG1）和核酸内切酶III同源物（NTH1）。我们发现OGG1在线粒体中氧化病变的修复中起着至关重要的作用，并且可能是在这些细胞器中去除8-oxog的唯一DNA糖基化酶。我们还表明，n位于线粒体中，它参与去除氧化的嘧啶。我们研究的一个重要点是，我们使用色谱技术测定DNA修复活性并实际发生DNA中病变的实际发生。我们分析了小鼠肝脏DNA中的8-氧化物和其他氧化碱的水平，发现环形开环的氧化病变fapyguanine（Fapyg）的水平高于8-oxog的水平。使用缺乏这些糖基化酶的小鼠模型，我们发现从OGG1 - / - 小鼠中积聚了8-oxog和Fapy-G，而Fapyg和Fapyadenine（Fapya）在NTH1 - / - 小鼠的DNA中积聚。我们还表明，Fapyg和Fapya通过相同的DNA糖基酶进行修复，这些DNA糖基化酶从核和线粒体中去除DNA的8-氧基和胸腺糖糖。这些结果表明，这些病变的积累可能至少与8-oxog一样重要。此外，我们确定了新鉴定的DNA糖基酶Neil1的线粒体定位，该糖基酶Neil1对环开环的底物具有更高的特异性。 在人类细胞中，表达两个不同的OGG1同工型，Alpha和beta。 β-OGG1仅本地定位于线粒体，据信提供8-氧化胶囊活性。我们纯化了重组B-OGG1，发现该蛋白缺乏糖基化酶活性。定点诱变研究鉴定出在B异型类型中发现的两种氨基酸，它们使A-异型不活跃。我们还发现，大约有10％的A-OGG1本地定位在线粒体上，并可能解释了线粒体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，因此我们正在研究它是否具有任何生物学功能。为此，我们正在用B-OGG1的同工型特异性稳定敲低建立细胞系，以确定在没有该蛋白质的情况下可能改变的可能生物终点。 所有BER酶均编码在细胞核中并运输到线粒体上。但是，有关线粒体BER的调节信息非常有限。我们测量了缺乏mtDNA（Rho-）的线粒体中的BER活性。尽管没有mtDNA，但与野生型线粒体相比，线粒体中存在完整的BER酶，大多数活性仅略有下降。有趣的是，核BER活性也受到mtDNA的缺乏的影响，这表明两个隔间中的BER之间都有一个有趣的串扰。线粒体由两个膜（外部和内部）组成，它们封闭了一个水基质室。我们研究了BER在线粒体中的空间组织，发现大多数BER活动不是在矩阵中自由溶解，而是与膜分数相关的。这种关联本质上可能是静电，因为它可能会被高盐浓度所破坏。这种高阶DNA修复复合物的存在对mtDNA修复具有深远的影响，因为它表明了一种机制，其中DNA流过这种固定复合物。在哺乳动物线粒体中，在称为核苷的大蛋白-DNA复合物中发现mtDNA。哺乳动物核苷的最丰富的蛋白质成分之一是转录因子TFAM，它被认为在压实mtDNA的核苷中具有结构功能。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与含有氧化碱基的DNA的亲和力更高，并且当TFAM结合时，BER酶的催化活性会降低，这很可能是由于对受损碱基的可及性较差。这些结果表明，TFAM可以通过翻译后修饰来调节BER，从而改变其DNA结合亲和力。 我们现在正在研究哺乳动物线粒体是否具有在细胞核中运行的其他任何修复途径，例如不匹配修复（MMR）。我们的结果表明，人线粒体可以在体外催化不匹配的修复并包含不匹配的结合活性。使用具有不匹配的DNA底物的亲和力纯化和质谱肽分析，我们在不匹配结合的复合物，转录因子YB-1，毒酶氧化酶 - 成分因子LRP130和一个未知活性的UV抗性基因的基因中鉴定了3种蛋白。我们使用内源性和异位表达的蛋白显示了YB-1的线粒体定位。有趣的是，在体外测定中，通过RNA干扰通过RNA干扰清除YB1水平显着降低了线粒体触及的错配修复活性，这表明该蛋白参与线粒体的MMR。这些观察结果以及其他观察结果清楚地表明，哺乳动物线粒体具有功能不匹配的修复途径。 线粒体DNA代谢涉及的另一组重要组合是Helicases SUV3和PIF1。我们已经研究了SUV3的生化功能，并且它似乎与某些线粒体和端粒蛋白相互作用，从而使其在端粒和线粒体中都具有功能。 这是进一步调查的。

项目成果

The mitochondrial theory of aging: involvement of mitochondrial DNA damage and repair.

• DOI: 10.1016/s0074-7742(02)53018-x
• 发表时间: 2002
• 期刊: International review of neurobiology
• 作者: N. C. de Souza-Pinto;V. Bohr

Localization of mitochondrial DNA base excision repair to an inner membrane-associated particulate fraction.

• DOI: 10.1093/nar/gki683
• 发表时间: 2005
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Stuart, JA;Mayard, S;Hashiguchi, K;Souza-Pinto, NC;Bohr, VA
• 通讯作者: Bohr, VA

Base excision repair in nuclear and mitochondrial DNA.

• DOI: 10.1016/s0079-6603(01)68107-8
• 发表时间: 2001
• 期刊: Progress in nucleic acid research and molecular biology
• 作者: G. Dianov;N. Souza-Pinto;S. Nyaga;Tanja Thybo;T. Stevnsner;V. Bohr

Hippocampal atrophy in the healthy is initially linear and independent of age.

健康人的海马萎缩最初是线性的并且与年龄无关。

• DOI: 10.1016/j.neurobiolaging.2005.07.018
• 发表时间: 2006
• 期刊: Neurobiology of aging
• 影响因子: 4.2
• 作者: Cohen,RobertM;Szczepanik,Joanna;McManus,Michael;Mirza,Nadeem;Putnam,Karen;Levy,Jim;Sunderland,Trey
• 通讯作者: Sunderland,Trey

Gene-specific and mitochondrial repair of oxidative DNA damage.

DNA 氧化损伤的基因特异性和线粒体修复。

• DOI: 10.1385/1-59259-973-7:155
• 发表时间: 2006
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Anson,RMichael;Mason,PenelopeA;Bohr,VilhelmA
• 通讯作者: Bohr,VilhelmA