DNA Repair And Somatic Mutation In Antibody Variable Gen

工作总结：线粒体DNA(MtDNA)由于靠近电子传递链而积累了高度的氧化损伤，大多数活性氧物种都是在电子传递链上产生的。氧化损伤会导致突变、缺失，并导致细胞死亡。8-羟基鸟嘌呤是一种主要的DNA氧化性损伤，随着年龄的增长在线粒体DNA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移除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糖基酶1(Ogg1)在线粒体DNA修复中的作用。我们的结果表明，Ogg1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8OHdG糖基酶。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，被认为提供8-OHdG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，并可能提供8-OHdG糖基酶活性。N TH是修复DNA氧化损伤的另一种主要糖基酶。我们研究了缺乏这种酶的小鼠肝脏线粒体的DNA修复。我们发现，这些线粒体提取物不能修复DNA中的胸腺嘧啶二醇损伤，表明第n个酶负责修复DNA中的这些损伤。我们观察到第n个基因敲打小鼠的提取物中其他氧化的嘧啶类化合物的残余切割活性，表明线粒体中可能存在一些微小的备用路径来移除这些损伤。这些DNA糖基酶在细胞核中编码，并运输到线粒体；然而，关于线粒体BER的调节信息非常有限。我们测量了缺乏线粒体DNA(Rho-)的线粒体的BER活性。尽管没有线粒体DNA，但存在完整的线粒体BER，与wt线粒体相比，大多数活性仅略有下降。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。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行的修复途径，例如错配修复。P53蛋白最近被认为与核内的误码率有关。我们研究了P53是否参与了线粒体的BER，发现从P53基因缺失的小鼠肝脏中提取的线粒体具有正常水平的DNA糖基酶和AP内切酶活性。DNA修复合成参入率略有下降，但可被重组P53刺激。在缺口填充试验中测量的DNA聚合酶伽马活性在基因敲除小鼠的提取物中也降低了。我们的结果表明，P53参与了线粒体BER中的核苷酸掺入步骤。卡路里限制(CR)是迄今为止唯一已知的延缓衰老的干预措施。我们研究了热量限制小鼠线粒体和细胞核中的DNA修复活性，以确定DNA修复是否受到此类饮食变化的影响。我们发现CR对核和线粒体的误码率有不同的调节作用。在CR动物中，核BER显着上调，而线粒体BER仅略高。我们还观察了不同器官对CR反应的差异。我们的结果表明，在CR过程中，BER没有发生普遍的上调。我们研究的一个优点是我们检测DNA修复活性，并使用高效液相色谱法和其他分析技术测量DNA中损伤的实际发生情况。

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.