DNA Repair And Somatic Mutation In Antibody Variable Gen

抗体可变基因中的 DNA 修复和体细胞突变

• 批准号: 6969397
• 负责人: Vilhelm A Bohr
• 金额: --
• 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/6969397
• 关键词: DNA repair; N glycosidase; aging; enzyme activity; genetically modified animals; high performance liquid chromatography; laboratory mouse; mitochondrial DNA; oxidative stress; p53 gene /protein

Summary of work: The mitochondrial DNA (mtDNA) accumulates high levels of oxidative damage owing to its proximity to the electron transport chain, where most reactive oxygen species are generated. Oxidative damage can cause mutations, deletions and lead to cell death. 8-hydroxyguanine, a major oxidative DNA lesion, accumulates with age in the mtDNA. Repair of oxidative DNA damage is carried out by the base excision repair (BER) system. We investigate the repair mechanisms for the removal of DNA lesions, particularly oxidized bases, from the mitochondrial genome. We have investigated the role of the oxoguanine DNA glycosylase 1 (Ogg1) in mtDNA repair in mice that are defective in this enzyme. Our results suggest that Ogg1 plays a crucial role in the repair of oxidative damage in mitochondria and is probably the only 8OHdG glycosylase in these organelles. In human cells two distinct Ogg1 isoforms are expressed, alpha and beta. Beta-Ogg1 localizes only to mitochondria and was believed to provide the 8-OHdG 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 provide the 8-OHdG glycosylase activity. NTH is the other major glycosylase for repair of oxidative DNA damage. We investigated DNA repair in liver mitochondria from mice deficient in this enzyme. We found that those mitochondrial extracts can not repair thymine glycol lesions in DNA, indicating that the NTH enzyme is responsible for the repair of these lesions in DNA. We observed some residual incision acitvity for other oxidized pyrimidines in extracts from NTH knockou mice, suggesting that some minor backup pathways my exist in mitochondria for the removal of these lesions. These DNA glycosylases 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 mitochondrial BER was present, and most activities were only slightly decreased compared to wt mitochondria. Interestingly, nuclear BER activities were also affected by the absence of mtDNA, suggesting an interesting cross-talk between BER in both compartments. We are now investigating whether mammalian mitochondria have other repair pathways that operate in the nucleus, such as mismatch repair. The p53 protein has recently been associated with BER in the nucleus. We investigated whether p53 participates in BER in mitochondria and found that mitochondrial extracts from p53 null mouse liver have normal levels of DNA glycosylase and AP-endonulcease activities. DNA repair synthesis incorporation is slightly decrease but can be stimulated by addition of recombinant p53. DNA polymerase gamma activity, measured in a gap-filling assay, was also decreased in extracts from the knockout mice. Our results suggest that p53 participates in the nucleotide incorporation step in mitochondrial BER. Calorie restriction (CR) is the only intervention known so far that slows aging. We studied DNA repair activities in mitochondriaand nuclei from caloric restricted mice to determine whether DNA repair is affected by such dietary changes. We found that CR modulates nuclear and mitochondrial BER differentially. Nuclear BER is significantly up-regulated in CR animals while mitochondria BER is only slightly higher. We also observed organ specific differences in the response to CR. Our results indicate that a general up-regulation of BER does not occur during CR. One strenght of our studies is that we assay for DNA repair activity and measure the actual occurrence of the lesions in DNA using HPLC and other analytical techniques.

工作总结：线粒体DNA（mtDNA）由于其接近电子传递链而积累高水平的氧化损伤，其中产生大多数活性氧。氧化损伤可引起突变、缺失并导致细胞死亡。8-羟基鸟嘌呤是一种主要的DNA氧化损伤，随着年龄的增长在mtDNA中积累。DNA氧化损伤的修复是通过碱基切除修复（BER）系统进行的。我们研究了从线粒体基因组中去除DNA损伤，特别是氧化碱基的修复机制。我们研究了氧鸟嘌呤DNA糖基化酶1（Ogg 1）在这种酶缺陷的小鼠mtDNA修复中的作用。我们的研究结果表明，Ogg 1在线粒体氧化损伤的修复中起着至关重要的作用，可能是这些细胞器中唯一的8 OHdG糖基化酶。在人类细胞中，表达两种不同的Ogg 1亚型，α和β。Beta-Ogg 1仅定位于线粒体，并被认为提供8-OHdG糖基化酶活性。我们纯化了重组b-Ogg 1，发现该蛋白缺乏糖基化酶活性。定点诱变研究鉴定了在b-亚型中发现的使a-亚型失活的两个氨基酸。我们还发现，约10%的a-Ogg 1定位于线粒体，并可能提供8-OHdG糖基化酶活性。NTH是用于修复氧化DNA损伤的另一种主要糖基化酶。我们研究了缺乏这种酶的小鼠肝脏线粒体中的DNA修复。我们发现这些线粒体提取物不能修复胸腺嘧啶乙二醇损伤的DNA，表明NTH酶负责修复这些DNA损伤。我们在NTH敲除小鼠的提取物中观察到其他氧化嘧啶的一些残留切割活性，这表明线粒体中可能存在一些次要的备用途径来去除这些病变。这些DNA糖基化酶在细胞核中编码并转运到线粒体;然而，关于线粒体BER的调控的信息非常有限。我们测量了BER活动的线粒体，缺乏mtDNA（rho-）。尽管没有线粒体DNA的完整的线粒体BER是存在的，和大多数活动相比，野生型线粒体仅略有下降。有趣的是，核BER活动也受到mtDNA缺失的影响，这表明两个隔室中BER之间存在有趣的串扰。我们现在正在研究哺乳动物线粒体是否有其他在细胞核中运作的修复途径，如错配修复。最近，p53蛋白与细胞核中的BER相关。我们研究了p53是否参与线粒体BER，发现p53缺失小鼠肝脏线粒体提取物具有正常水平的DNA糖基化酶和AP-内切酶活性。DNA修复合成掺入略有减少，但可通过添加重组p53来刺激。DNA聚合酶γ活性，在间隙填充试验中测量，也减少了基因敲除小鼠的提取物。我们的研究结果表明，p53参与线粒体BER的核苷酸掺入步骤。卡路里限制（CR）是迄今为止已知的唯一延缓衰老的干预措施。我们研究了热量限制小鼠的睾丸和细胞核中的DNA修复活性，以确定DNA修复是否受到这种饮食变化的影响。我们发现，CR调制核和线粒体BER差异。核BER在CR动物中显著上调，而线粒体BER仅略高。我们还观察到对CR反应的器官特异性差异。我们的研究结果表明，一般上调的BER不会发生在CR。我们的研究的一个优势是我们检测DNA修复活性，并使用HPLC和其他分析技术测量DNA中损伤的实际发生。