Mitochondrial DNA Repair In Oxidative Stress And Aging

工作总结：我们已经报道，有一个线粒体DNA糖基化酶/核酸内切酶，识别病变8-OH-脱氧鸟苷（8-OHdG）的活性随着年龄的增长。这种酶的活性增加，从6至23个月的年龄在大鼠和小鼠的肝脏线粒体提取物。与此相反，其他两个线粒体酶的DNA代谢，这不是专门参与修复的氧化损伤，尿嘧啶DNA糖基化酶（mtUDG）和AP核酸内切酶，没有变化的活动与老化。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化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糖基化酶1（OGG 1）在这种酶缺陷的小鼠mtDNA修复中的作用。我们发现OGG 1基因敲除小鼠的肝线粒体没有可检测到的8 OHdG切割活性，表明线粒体活性是由与核酶相同的基因编码的。来自敲除小鼠的线粒体DNA积累的8 OHdG比野生型动物多9倍。相比之下，来自相同动物的核DNA的8 OHdG修饰仅比对照多两倍。这些结果表明，OGG 1在线粒体氧化损伤的修复中起着至关重要的作用，并且可能是这些细胞器中唯一的8 OHdG糖基化酶。NTH是另一种修复氧化DNA损伤的糖基化酶。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氧化损伤的修复是通过碱基切除修复（BER）系统进行的。BER可以通过两种途径发生，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修复补丁的大小。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同一损伤的核修复。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糖基化酶活性和DNA修复合成掺入。然而，γ射线照射后，线粒体内的p53和修复合成水平略有升高，这表明p53易位到线粒体可能会调节BER上调响应于应力。为了研究BER的组织特异性，我们测定了小鼠睾丸、肝、肾、肌肉、脑和心脏的核和线粒体提取物中的DNA糖基化酶活性。睾丸细胞核和线粒体中的BER水平最高，这表明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，BER水平在不同的器官之间差异很大。热量限制是一种主要的治疗干预对年龄相关的退化。我们正在研究热量限制小鼠的DNA氧化损伤的DNA修复，以确定DNA修复是否受到这种饮食变化的影响。我们还对特定DNA修复基因缺陷的敲除小鼠进行热量限制。这是确定DNA修复是否在此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中损伤的实际发生。

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.