Mitochondrial DNA Repair In Oxidative Stress And Aging

工作总结：我们已经报道了线粒体DNA糖基酶/内切酶的活性随着年龄的增长而增加，该酶可以识别损伤的8- oh脱氧鸟苷（8-OHdG）。在大鼠和小鼠的肝脏线粒体提取物中，该酶的活性在6 ~ 23月龄时增加。相反，另外两种DNA代谢的线粒体酶，尿嘧啶DNA糖基化酶（mtUDG）和AP内切酶，不特异性参与氧化损伤的修复，随着年龄的增长，活性没有变化。在小鼠肝脏中，我们观察到线粒体DNA的DNA修复随着年龄的增长而增加，而核DNA修复随着年龄的增长而略有下降。线粒体碱基切除修复糖基酶很可能是由随着年龄增长而积累的DNA损伤诱导的。我们还研究了氧鸟嘌呤DNA糖基酶1 （OGG1）在小鼠mtDNA修复中的作用，该酶存在缺陷。我们发现，OGG1基因敲除小鼠的肝脏线粒体没有可检测到的8OHdG切口活性，这表明线粒体活性与核酶是由同一基因编码的。基因敲除小鼠的线粒体DNA积累的8OHdG是正常小鼠的9倍。相比之下，来自同一动物的核DNA的8OHdG修饰仅比对照多两倍。这些结果表明，OGG1在线粒体氧化损伤的修复中起着至关重要的作用，并且可能是这些细胞器中唯一的8OHdG糖基酶。NTH是另一种修复DNA氧化损伤的糖基酶。我们研究了缺乏这种酶的小鼠肝脏线粒体的DNA修复。我们发现这些细胞提取物不能修复DNA中的胸腺嘧啶乙二醇损伤，表明NTH酶负责修复DNA中的这些损伤。DNA氧化损伤的修复是由碱基切除修复（BER）系统进行的。BER可通过两种途径发生，即长补丁修复和短补丁修复。我们研究了人细胞线粒体中尿嘧啶修复过程中的修复斑块大小。我们的研究结果表明，DNA中的尿嘧啶仅通过线粒体中的短补丁途径修复，而长补丁途径广泛用于相同病变的核修复。最近发现p53蛋白与细胞核内的BER有关。我们研究了p53是否参与线粒体BER，发现p53缺失小鼠肝脏的线粒体提取物具有正常水平的DNA糖基酶活性和DNA修复合成整合。然而，伽马辐照后，线粒体内p53水平和修复合成水平略有升高，这表明p53易位到线粒体可能调节了应激反应中BER的上调。为了研究BER的组织特异性，我们测量了小鼠睾丸、肝脏、肾脏、肌肉、大脑和心脏的核和线粒体提取物的DNA糖基酶活性。睾丸细胞核和线粒体的BER水平最高，表明BER在维持遗传完整性方面起着关键作用。我们的研究结果表明，不同器官的BER水平差异很大。热量限制是对抗年龄相关变性的主要治疗干预措施。我们正在研究热量限制小鼠氧化DNA损伤的DNA修复，以确定DNA修复是否受到这种饮食变化的影响。我们还对基因敲除的老鼠进行了热量限制，这些老鼠在特定的DNA修复基因上有缺陷。这是确定DNA修复是否在这一过程中发挥作用的另一种方法。在我们的研究中，我们检测DNA修复活性，我们还使用HPLC和其他技术测量DNA损伤的实际发生情况。

Summary of work: We have reported that there is an increase with age in the activity of a mitochondrial DNA glycosylase/endonuclease that recognizes the lesion 8-OH-deoxyguanosine (8-OHdG). The activity of this enzyme increased from 6 to 23 months of age in liver mitochondrial extracts from rats and mice. In contrast, two other mitochondrial enzymes of DNA metabolism which are not specifically involved in the repair of oxidative damage, uracil DNA glycosylase (mtUDG) and AP endonuclease, had no change in activity with aging. In mouse liver, we observed an increase in DNA repair with age for the mitochondrial DNA, while the nuclear DNA repair slightly decreased with age. It is likely that the mitochondrial base excision repair glycosylases are induced by DNA damage that accumulates there with age. We have also investigated the role of the oxoguanine DNA glycosylase 1 (OGG1) in mtDNA repair in mice that are defective in this enzyme. We found that liver mitochondria from the OGG1 knockout mice have no detectable 8OHdG incision activity, demonstrating that the mitochondrial activity is encoded by the same gene as the nuclear enzyme. Mitochondrial DNA from the knockout mice accumulate 9 times more 8OHdG than wt animals. In contrast, nuclear DNA from the same animals have only two time more 8OHdG modifications than controls. These 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. NTH is another glycosylase that repairs oxidative DNA damage. We investigated DNA repair in liver mitochondria from mice deficient in this enzyme. We found that those cell extracts can not repair thymine glycol lesions in DNA, indicating that the NTH enzyme is responsible for the repair of these lesions in DNA. Repair of oxidative DNA damage is carried out by the base excision repair (BER) system. BER can occur through two pathways, long and short patch repair. We investigated the repair patch size during repair of uracil in mitochondria from human cells. Our results show that uracil in DNA is repaired solely by the short patch pathway in mitochondria, while the long patch pathway is extensively used in the nuclear repair of the same lesion. 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 activity and of DNA repair synthesis incorporation. However, after gamma irradiation, the intramitochondrial levels of p53 and repair synthesis are slightly elevated, suggesting that p53 translocation to mitochondria may modulate BER up-regulation in response to stress. To investigate the tissue specificity of BER we measured DNA glycosylase actvities in nuclear and mitochondrial extracts from mouse testis, liver, kidney, muscle, brain and heart. Testis had the highest BER levels in both nucleus and mitochondria, suggesting that BER plays a critical role in maintaining genetic integrity. Our results show that BER levels vary greatly among the different organs. Caloric restriction is a major therapeutic intervention against age associated degeneration. We are studying DNA repair of oxidative DNA lesions in calorically restricted mice to determine whether DNA repair is affected by such dietary changes. We are also subjecting knockout mice, defective in specific DNA repair genes, to caloric restriction. This is another approach to determine whether DNA repair plays a role in this process. In our studies, we assay for DNA repair activity, and we also measure the actual occurrence of the lesions in DNA using HPLC and other techniques.