Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells.
Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells.
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DOI：
10.1093/nar/gkl703
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发表时间：
2006
影响因子：
14.9
通讯作者：
Jacobs, Howard T.
中科院分区：
文献类型：
--
作者：
Pohjoismaki, Jaakko L. O.;Wanrooij, Sjoerd;Hyvarinen, Anne K.;Goffart, Steffi;Holt, Ian J.;Spelbrink, Johannes N.;Jacobs, Howard T.
线粒体转录因子A（TFAM）是HMG超家族中一种丰富的线粒体蛋白，在线粒体DNA（mtDNA）代谢中具有多种推测的作用。在这项研究中，我们研究了在培养的人类细胞中操纵TFAM表达对mtDNA复制的影响。哺乳动物线粒体DNA复制中间体（RIs）分为两类，它们之间的相互作用机制还不清楚.一类的特点是广泛的RNA掺入的滞后链，而另一种具有传统的，链偶联复制的产品的结构。TFAM过表达增加了RI的总体丰度，并将其基本上转向传统的链偶联型。这种转变在rDNA区域和各种复制暂停位点最为明显，并伴随着复制终止中间体相对量的下降、线粒体转录本的大幅减少、mtDNA脱链和进行性拷贝数耗竭。TFAM过表达可以通过用双脱氧胞苷处理细胞来部分表型化，这表明其作用部分归因于分叉进展速率降低。TFAM敲低也导致mtDNA耗竭，但RI仍然主要是核糖取代型，虽然终止中间体增强。我们认为TFAM通过对mtDNA代谢不同方面的综合作用影响mtDNA的复制模式。
Mitochondrial transcription factor A (TFAM) is an abundant mitochondrial protein of the HMG superfamily, with various putative roles in mitochondrial DNA (mtDNA) metabolism. In this study we have investigated the effects on mtDNA replication of manipulating TFAM expression in cultured human cells. Mammalian mtDNA replication intermediates (RIs) fall into two classes, whose mechanistic relationship is not properly understood. One class is characterized by extensive RNA incorporation on the lagging strand, whereas the other has the structure of products of conventional, strand-coupled replication. TFAM overexpression increased the overall abundance of RIs and shifted them substantially towards those of the conventional, strand-coupled type. The shift was most pronounced in the rDNA region and at various replication pause sites and was accompanied by a drop in the relative amount of replication-termination intermediates, a substantial reduction in mitochondrial transcripts, mtDNA decatenation and progressive copy number depletion. TFAM overexpression could be partially phenocopied by treatment of cells with dideoxycytidine, suggesting that its effects are partially attributable to a decreased rate of fork progression. TFAM knockdown also resulted in mtDNA depletion, but RIs remained mainly of the ribosubstituted type, although termination intermediates were enhanced. We propose that TFAM influences the mode of mtDNA replication via its combined effects on different aspects of mtDNA metabolism.
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