Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage.
Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage.
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DOI：
10.1371/journal.pgen.1003794
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Loeb LA
中科院分区：
文献类型：
--
作者：
Kennedy SR;Salk JJ;Schmitt MW;Loeb LA
线粒体DNA（mtDNA）被认为是非常容易受到年龄相关的损伤和活性氧（ROS）的诱变。然而，体细胞mtDNA突变历来难以研究，因为在精确定量罕见mtDNA突变方面存在技术限制。我们应用了高灵敏度的双链测序方法，该方法可以在>107个野生型分子中检测单个突变，以前所未有的准确度对从年轻和老年人的人脑组织中纯化的mtDNA进行测序。我们发现，在80岁的生命过程中，点突变的频率增加了105倍。总体而言，两组的突变谱主要由过渡突变组成，与DNA聚合酶γ的错误掺入或胞苷和腺苷的脱氨基作用作为mtDNA中的主要致突变事件一致。令人惊讶的是，被认为是DNA氧化损伤标志的G→T突变并没有随着年龄的增长而显著增加。我们观察到一个不均匀的，与年龄无关的mtDNA突变分布，与D-环表现出显着较高的突变频率比基因组的其余部分。编码区（而不是D环）在两条链之间表现出明显的突变不对称积累，G→A和T→C突变更常发生在轻链上而不是重链上。我们在数据中观察到的模式和偏差密切反映了人类种群和密切相关物种研究中报告的突变谱。总的来说，我们的研究结果反对氧化损伤是衰老的主要驱动因素，并表明DNA聚合酶γ和/或自发碱水解的复制错误是导致mtDNA中大量累积点突变的原因。由于它们的进化历史，线粒体拥有独立复制的基因组。在复制过程中不能忠实地传递线粒体DNA的遗传信息会导致功能失调的电子传递蛋白的产生和随后的能量产生下降。与核DNA相比，细胞来源的活性氧（ROS）和环境因子优先损伤mtDNA。然而，很少有人知道的后果mtDNA损伤的诱变。这种知识的缺乏部分源于缺乏能够在整个线粒体基因组中准确检测这些突变的方法。使用一种新的、高度敏感的DNA测序策略，我们发现点突变的频率比以前使用不太精确的方法报道的低10-100倍。此外，在80年的寿命中，频率增加了5倍。我们还发现，随着年龄的增长，主要是过渡突变，而不是通常与mtDNA氧化损伤相关的突变。这一发现与衰老的自由基理论不一致。最后，我们在数据中观察到的致突变模式和偏差与线粒体多态性的人群研究中所见相似，并表明体细胞和种系mtDNA突变的共同机制。
Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >107 wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ∼5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G→T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G→A and T→C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA. Owing to their evolutionary history, mitochondria harbor independently replicating genomes. Failure to faithfully transmit the genetic information of mtDNA during replication can lead to the production of dysfunctional electron transport proteins and a subsequent decline in energy production. Cellularly-derived reactive oxygen species (ROS) and environmental agents preferentially damage mtDNA compared to nuclear DNA. However, little is known about the consequences of mtDNA damage for mutagenesis. This lack of knowledge stems, in part, from an absence of methods capable of accurately detecting these mutations throughout the mitochondrial genome. Using a new, highly sensitive DNA sequencing strategy, we find that the frequency of point mutations is 10–100-fold lower than what has been previously reported using less precise means. Moreover, the frequency increases 5-fold over an 80 year lifespan. We also find that it is predominantly transition mutations, rather than mutations commonly associated with oxidative damage to mtDNA, that increase with age. This finding is inconsistent with free radical theories of aging. Finally, the mutagenic patterns and biases we observe in our data are similar to what is seen in population studies of mitochondrial polymorphisms and suggest a common mechanism by which somatic and germline mtDNA mutations arise.
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影响因子：
4.5
作者：
Ameur A;Stewart JB;Freyer C;Hagström E;Ingman M;Larsson NG;Gyllensten U
通讯作者：
Gyllensten U
影响因子：
3.8
作者：
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Eide, Lars
DOI：
10.1111/j.1532-5415.1972.tb00787.x
发表时间：
1972-01-01
影响因子：
6.3
作者：
HARMAN, D
通讯作者：
HARMAN, D
人类结肠上皮和疾病中线粒体突变光谱的比较：缺乏在细胞线粒体DNA点突变中纯化选择的证据。
影响因子：
4.5
作者：
Greaves LC;Elson JL;Nooteboom M;Grady JP;Taylor GA;Taylor RW;Mathers JC;Kirkwood TB;Turnbull DM
通讯作者：
Turnbull DM
影响因子：
7
作者：
Galtier, N;Enard, D;Belkhir, K
通讯作者：
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