Mitochondrial DNA Repair Processes In Oxidative Stress And Aging

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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.