Role of Direct Repeat and Stem-Loop Motifs in mtDNA Deletions: Cause or Coincidence?

Deletion mutations within mitochondrial DNA (mtDNA) have been implicated in degenerative and aging related conditions, such as sarcopenia and neuro-degeneration. While the precise molecular mechanism of deletion formation in mtDNA is still not completely understood, genome motifs such as direct repeat (DR) and stem-loop (SL) have been observed in the neighborhood of deletion breakpoints and thus have been postulated to take part in mutagenesis. In this study, we have analyzed the mitochondrial genomes from four different mammals: human, rhesus monkey, mouse and rat, and compared them to randomly generated sequences to further elucidate the role of direct repeat and stem-loop motifs in aging associated mtDNA deletions. Our analysis revealed that in the four species, DR and SL structures are abundant and that their distributions in mtDNA are not statistically different from randomized sequences. However, the average distance between the reported age associated mtDNA breakpoints and their respective nearest DR motifs is significantly shorter than what is expected of random chance in human (p<10−4) and rhesus monkey (p = 0.0034), but not in mouse (p = 0.0719) and rat (p = 0.0437), indicating the existence of species specific difference in the relationship between DR motifs and deletion breakpoints. In addition, the frequencies of large DRs (>10 bp) tend to decrease with increasing lifespan among the four mammals studied here, further suggesting an evolutionary selection against stable mtDNA misalignments associated with long DRs in long-living animals. In contrast to the results on DR, the probability of finding SL motifs near a deletion breakpoint does not differ from random in any of the four mtDNA sequences considered. Taken together, the findings in this study give support for the importance of stable mtDNA misalignments, aided by long DRs, as a major mechanism of deletion formation in long-living, but not in short-living mammals.

线粒体DNA（mitochondrial DNA, mtDNA）内的缺失突变已被证实与退行性疾病及衰老相关病症存在关联，例如肌肉减少症（sarcopenia）与神经退行性病变。尽管mtDNA缺失形成的确切分子机制尚未完全阐明，但已有研究在缺失断裂点附近观察到同向重复序列（direct repeat, DR）与茎环结构（stem-loop, SL）等基因组基序，因此推测二者可能参与了诱变过程。本研究对人类、恒河猴、小鼠与大鼠四种不同哺乳动物的线粒体基因组进行了分析，并将其与随机生成的序列进行比对，以进一步阐明同向重复序列与茎环结构在衰老相关mtDNA缺失中的作用。分析结果显示，在这四个物种中，同向重复序列与茎环结构的丰度较高，且它们在线粒体DNA中的分布与随机序列无统计学差异。然而，在人类（p<10⁻⁴）与恒河猴（p=0.0034）中，已报道的衰老相关mtDNA断裂点与其最近的同向重复序列基序之间的平均距离显著短于随机预期；但在小鼠（p=0.0719）与大鼠（p=0.0437）中则无此现象，这表明同向重复序列基序与缺失断裂点之间的关联存在物种特异性差异。此外，在所研究的四种哺乳动物中，长同向重复序列（长度>10 bp）的频率随寿命延长呈下降趋势，这进一步提示在长寿动物中，存在针对由长同向重复序列介导的不稳定线粒体DNA错配的进化选择压力。与同向重复序列的研究结果相反，在本次分析的四种线粒体DNA序列中，缺失断裂点附近出现茎环结构基序的概率与随机分布并无显著差异。综上，本研究结果支持：由长同向重复序列介导的稳定线粒体DNA错配，是长寿哺乳动物mtDNA缺失形成的主要机制，但这一机制并不适用于短寿哺乳动物。