Mitochondrial DNA Repair Processes In Oxidative Stress And Aging

碱基切除修复途径是由一类被称为DNA糖基酶的酶的作用启动的，这些酶识别并释放受损的碱基，从而赋予修复过程特异性。哺乳动物细胞携带两种主要用于修复氧化碱基的DNA糖基酶，氧鸟嘌呤DNA糖基酶（OGG1）和内切酶III同源物（NTH1）。我们发现，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去除8-oxoG的DNA糖基酶。在人类细胞中表达两种不同的OGG1亚型，α和β。所有的BER酶都在细胞核中编码并转运到线粒体；然而，关于线粒体BER调控的信息非常有限。在哺乳动物的线粒体中，线粒体dna存在于被称为类核的大型蛋白质- dna复合体中。转录因子TFAM是哺乳动物类核中最丰富的蛋白质成分之一，它被认为在将mtDNA压缩成类核结构方面具有结构功能。利用重组人TFAM，我们研究了TFAM是否可以调节mtDNA修复。我们发现TFAM可以抑制BER蛋白和线粒体pol γ。为了探究TFAM对活性的抑制是否与TFAM高亲和力DNA结合有关，我们创建了一个TFAM DNA结合突变体。我们观察到含有TFAM DNA结合突变体的反应受到较少的抑制。我们提出TFAM可能像核组蛋白一样起作用，因此提出TFAM重塑蛋白必须在线粒体中退出以允许mtDNA代谢。我们继续证明p53，一种已知的TFAM相互作用蛋白，可以缓解TFAM对OGG1切口的抑制。我们正在继续寻找和询问蛋白质与TFAM的相互作用，试图更全面地表征mtDNA修复和代谢。

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. In human cells two distinct OGG1 isoforms are expressed, alpha and beta. All BER enzymes are encoded in the nucleus and transported to mitochondria; however there is very limited information on the regulation of mitochondrial BER. 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 mtDNA into the nucleoid structure. Using recombinant human TFAM, we investigated whether TFAM could modulate mtDNA repair. We find that TFAM could inhibit BER proteins and mitochondrial pol gamma. To explore whether this inhibition of activity by TFAM was a function of TFAMs high affinity DNA binding we created a TFAM DNA binding mutant. We observed less inhibition in those reactions containing the DNA binding mutant of TFAM. We proposed that TFAM may be functioning like nuclear histones and therefore proposed that a TFAM remodeling protein must exit in mitochondria to allow for mtDNA metabolism. We went on to show that p53, a known TFAM interacting protein, could relieve TFAM inhibition of OGG1 incision. We are continuing to search for and interrogate protein-interaction with TFAM in an attempt to more fully characterize mtDNA repair and metabolism. 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.