Kozhukhar Natalya, Spadafora Domenico, Fayzulin Rafik, Shokolenko Inna N, Alexeyev Mikhail
a Department of Physiology and Cell Biology , University of South Alabama , Mobile , AL , USA.
b Department of Pharmacology , University of South Alabama , Mobile , AL , USA and.
Mitochondrial DNA A DNA Mapp Seq Anal. 2016 Nov;27(6):4390-4396. doi: 10.3109/19401736.2015.1089539. Epub 2015 Oct 16.
Translesion synthesis by specialized DNA polymerases is an important strategy for mitigating DNA damage that cannot be otherwise repaired either due to the chemical nature of the lesion. Apurinic/Apyrimidinic (abasic, AP) sites represent a block to both transcription and replication, and are normally repaired by the base excision repair (BER) pathway. However, when the number of abasic sites exceeds BER capacity, mitochondrial DNA is targeted for degradation. Here, we used two uracil-N-glycosylase (UNG1) mutants, Y147A or N204D, to generate AP sites directly in the mtDNA of NIH3T3 cells in vivo at sites normally occupied by T or C residues, respectively, and to study repair of these lesions in their native context. We conclude that mitochondrial DNA polymerase γ (Pol γ) is capable of translesion synthesis across AP sites in mitochondria of the NIH3T3 cells, and obeys the A-rule. However, in our system, base excision repair (BER) and mtDNA degradation occur more frequently than translesion bypass of AP sites.
由特殊DNA聚合酶进行的跨损伤合成是减轻因损伤的化学性质而无法以其他方式修复的DNA损伤的重要策略。无嘌呤/无嘧啶（脱碱基，AP）位点对转录和复制均构成障碍，通常由碱基切除修复（BER）途径进行修复。然而，当脱碱基位点的数量超过BER能力时，线粒体DNA会被靶向降解。在此，我们使用了两个尿嘧啶-N-糖基化酶（UNG1）突变体Y147A或N204D，分别在体内NIH3T3细胞的线粒体DNA中通常被T或C残基占据的位点直接产生AP位点，并在其天然环境中研究这些损伤的修复情况。我们得出结论，线粒体DNA聚合酶γ（Pol γ）能够在NIH3T3细胞线粒体中的AP位点上进行跨损伤合成，并遵循A规则。然而，在我们的系统中，碱基切除修复（BER）和线粒体DNA降解比AP位点的跨损伤绕过更频繁地发生。
