Gureev Artem P, Shaforostova Ekaterina A, Starkov Anatoly A, Popov Vasily N
Department of Genetics, Cytology and Bioengineering, Voronezh State University, Voronezh, Russia.
Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, NY, USA.
Toxicology. 2017 May 1;382:67-74. doi: 10.1016/j.tox.2017.03.010. Epub 2017 Mar 9.
Damage to mitochondrial DNA (mtDNA) is a meaningful biomarker for evaluating genotoxicity of drugs and environmental toxins. Existing PCR methods utilize long mtDNA fragments (∼8-10kb), which complicates detecting exact sites of mtDNA damage. To identify the mtDNA regions most susceptible to damage, we have developed and validated a set of primers to amplify ∼2kb long fragments, while covering over 95% of mouse mtDNA. We have modified the detection method by greatly increasing the enrichment of mtDNA, which allows us solving the problem of non-specific primer annealing to nuclear DNA. To validate our approach, we have determined the most damage-susceptible mtDNA regions in mice treated in vivo and in vitro with rotenone and HO. The GTGR-sequence-enriched mtDNA segments located in the D-loop region were found to be especially susceptible to damage. Further, we demonstrate that HO-induced mtDNA damage facilitates the relaxation of mtDNA supercoiled conformation, making the sequences with minimal damage more accessible to DNA polymerase, which, in turn, results in a decrease in threshold cycle value. Overall, our modified PCR method is simpler and more selective to the specific sites of damage in mtDNA.
线粒体DNA（mtDNA）损伤是评估药物和环境毒素遗传毒性的一个有意义的生物标志物。现有的聚合酶链式反应（PCR）方法利用的是较长的mtDNA片段（约8 - 10kb），这使得检测mtDNA损伤的确切位点变得复杂。为了确定mtDNA中最易受损伤的区域，我们开发并验证了一组引物，用于扩增约2kb长的片段，同时覆盖超过95%的小鼠mtDNA。我们通过大幅提高mtDNA的富集度对检测方法进行了改进，这使我们能够解决引物与核DNA非特异性退火的问题。为了验证我们的方法，我们确定了在体内和体外用鱼藤酮和羟基自由基（HO）处理的小鼠中最易受损伤的mtDNA区域。位于D环区域的富含GTGR序列的mtDNA片段被发现特别容易受到损伤。此外，我们证明HO诱导的mtDNA损伤促进了mtDNA超螺旋构象的松弛，使损伤最小的序列更容易被DNA聚合酶识别，进而导致阈值循环值降低。总体而言，我们改进的PCR方法对mtDNA损伤的特定位点更简单且更具选择性。
