MitoRCA-seq reveals unbalanced cytocine to thymine transition in Polg mutant mice.
MitoRCA-seq reveals unbalanced cytocine to thymine transition in Polg mutant mice.
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MitoRCA-seq 揭示了 Polg 突变小鼠中胞嘧啶向胸腺嘧啶转变的不平衡。
DOI：
10.1038/srep12049
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发表时间：
2015-07-27
影响因子：
4.6
通讯作者：
Zhu J
中科院分区：
文献类型：
--
作者：
Ni T;Wei G;Shen T;Han M;Lian Y;Fu H;Luo Y;Yang Y;Liu J;Wakabayashi Y;Li Z;Finkel T;Xu H;Zhu J
线粒体DNA （mtDNA）的突变可导致广泛的人类疾病。我们已经开发了一种深度测序策略，mitoRCA-seq，以检测低频mtDNA点突变，从总DNA的1ng开始。它采用滚圈扩增，通过定制的mtDNA特异性引物或商业试剂盒丰富全长圆形mtDNA，并最大限度地减少核编码线粒体DNA （Numts）的污染。通过分析野生型和Polg（线粒体DNA聚合酶γ）突变小鼠的突变谱，我们发现校对缺陷mtDNA聚合酶的小鼠通过增加突变位点的数量来显著增加突变负荷，并且在较小程度上通过提高现有位点的突变频率来降低突变负荷，甚至在早衰表型出现之前。引人注目的是，细胞素(C)到胸腺嘧啶(T)的转换在Polg突变小鼠的mtDNA中被发现过多。与其他类型的突变相比，C→T转变倾向于增加基础氨基酸的疏水性，并可能导致Polg突变小鼠的蛋白质功能受损。综上所述，我们的发现可能为进一步研究Polg突变小鼠早衰表型的分子机制提供线索。
Mutations in mitochondrial DNA (mtDNA) can lead to a wide range of human diseases. We have developed a deep sequencing strategy, mitoRCA-seq, to detect low-frequency mtDNA point mutations starting with as little as 1 ng of total DNA. It employs rolling circle amplification, which enriches the full-length circular mtDNA by either custom mtDNA-specific primers or a commercial kit, and minimizes the contamination of nuclear encoded mitochondrial DNA (Numts). By analyzing the mutation profiles of wild-type and Polg (mitochondrial DNA polymerase γ) mutant mice, we found that mice with the proofreading deficient mtDNA polymerase have a significantly higher mutation load by expanding the number of mutation sites and to a lesser extent by elevating the mutation frequency at existing sites even before the premature aging phenotypes appear. Strikingly, cytocine (C) to thymine (T) transitions are found to be overrepresented in the mtDNA of Polg mutated mice. The C → T transition, compared to other types of mutations, tends to increase the hydrophobicity of the underlying amino acids, and may contribute to the impaired protein function of the Polg mutant mice. Taken together, our findings may provide clues to further investigate the molecular mechanism underlying premature aging phenotype in Polg mutant mice.