Biomedicum Helsinki, Research Program of Molecular Neurology, University of Helsinki, and Department of Neurology, Helsinki University Central Hospital, Helsinki, Finland.
Hum Mol Genet. 2010 Jul 1;19(13):2695-705. doi: 10.1093/hmg/ddq163. Epub 2010 Apr 22.
Mitochondrial DNA (mtDNA) is an essential multicopy genome, compacted into protein-DNA clusters called nucleoids. Maintaining an adequate mtDNA copy number is crucial for cellular viability. Loss of mtDNA results in severe human syndromes, whereas increased mtDNA copy number has been suggested to improve survival from myocardial infarction in mice and to be a promising therapeutic strategy for mitochondrial disease. The mechanisms that regulate mtDNA amount and organization are, however, not fully understood. Of the proteins required for mtDNA existence, only the mitochondrial helicase Twinkle and mitochondrial transcription factor A (TFAM) have been shown to increase mtDNA copy number in vivo, when expressed in physiological levels. Here we studied how Twinkle and TFAM affect mtDNA synthesis and nucleoid structure in mice. Using in vivo BrdU labeling, we show that Twinkle specifically regulates de novo mtDNA synthesis. Remarkably, high mtDNA copy number in mice is accompanied by nucleoid enlargement, which in turn correlates with defective transcription, age-related accumulation of mtDNA deletions and respiratory chain (RC) deficiency. Simultaneous overexpression of Twinkle and TFAM in bitransgenic mice has an additive effect on mtDNA copy number, increasing it up to 6-fold in skeletal muscle. Bitransgenic mice also exhibit further enlargement of nucleoids and aggravation of the RC defect. In conclusion, we show that Twinkle acts as a regulator of mtDNA replication initiation, and provide evidence that high mtDNA copy number and alteration of nucleoid architecture may be detrimental to mitochondrial function.
线粒体 DNA（mtDNA）是一种重要的多拷贝基因组，压缩成称为核体的蛋白质-DNA 簇。维持足够的 mtDNA 拷贝数对于细胞活力至关重要。mtDNA 的缺失会导致严重的人类综合征，而增加 mtDNA 拷贝数已被认为可以提高小鼠心肌梗死的存活率，并成为治疗线粒体疾病的有前途的策略。然而，调节 mtDNA 数量和组织的机制尚未完全理解。在需要存在 mtDNA 的蛋白质中，只有线粒体解旋酶 Twinkle 和线粒体转录因子 A（TFAM）在生理水平表达时，才被证明可以在体内增加 mtDNA 拷贝数。在这里，我们研究了 Twinkle 和 TFAM 如何影响小鼠的 mtDNA 合成和核体结构。通过体内 BrdU 标记，我们表明 Twinkle 特异性调节从头 mtDNA 合成。值得注意的是，小鼠中的高 mtDNA 拷贝数伴随着核体增大，这反过来又与转录缺陷、年龄相关的 mtDNA 缺失积累和呼吸链（RC）缺陷相关。在双转基因小鼠中同时过表达 Twinkle 和 TFAM 对 mtDNA 拷贝数具有加性效应，使骨骼肌中的 mtDNA 拷贝数增加高达 6 倍。双转基因小鼠还表现出核体进一步增大和 RC 缺陷加剧。总之，我们表明 Twinkle 作为 mtDNA 复制起始的调节剂起作用，并提供证据表明高 mtDNA 拷贝数和核体结构的改变可能对线粒体功能有害。
