Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation
Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation
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DOI：
10.1016/j.bbamcr.2010.04.008
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发表时间：
2010-08-01
影响因子：
5.1
通讯作者：
Yasukawa, Takehiro
中科院分区：
文献类型：
--
作者：
Ruhanen, Heini;Borrie, Sarah;Yasukawa, Takehiro
单链DNA结合蛋白(SSB)通过与单链DNA结合，在DNA复制、重组和修复中发挥重要作用。哺乳动物线粒体单链断裂(MtSSB)是细菌型单链断裂。在体外，mtSSB被证明能刺激线粒体复制DNA解旋酶和聚合酶的活性，但其在体内的功能尚未被详细研究。在这里，我们研究了mtSSB在维持培养的人类细胞线粒体DNA(MtDNA)中的作用。RNA干扰HeLa细胞中mtSSB的表达，导致该蛋白迅速减少，mtDNA拷贝数逐渐减少。HeLa细胞mtSSB基因敲除后，mtDNA合成率略有下降。这些结果证实了mtSSB对mtDNA复制的要求。哺乳动物线粒体DNA的许多分子都有一条短的第三链，即所谓的7S DNA，人们对其调控知之甚少。与mtDNA拷贝数逐渐减少相反，7S DNA在mtSSB基因敲除后在HeLa细胞中显著减少。此外，在骨肉瘤细胞系中，mtSSB基因敲除显著影响了7S DNA的合成。这些数据表明，mtSSB除了在mtDNA复制中发挥作用外，在7S DNA的维持中也起着重要作用。此外，线粒体DNA的活细胞染色并不意味着在HeLa细胞中线粒体核蛋白-mtDNA复合体在mtSSB被敲除时的组织结构发生变化。这一结果表明，7S DNA的存在对线粒体类核的组织并不是至关重要的。(C)2010爱思唯尔B.V.保留所有权利。
Single-stranded DNA binding protein (SSB) plays important roles in DNA replication, recombination and repair through binding to single-stranded DNA. The mammalian mitochondrial SSB (mtSSB) is a bacterial type SSB. In vitro, mtSSB was shown to stimulate the activity of the mitochondrial replicative DNA helicase and polymerase, but its in vivo function has not been investigated in detail. Here we studied the role of mtSSB in the maintenance of mitochondrial DNA (mtDNA) in cultured human cells. RNA interference of mtSSB expression in HeLa cells resulted in rapid reduction of the protein and a gradual decline of mtDNA copy number. The rate of mtDNA synthesis showed a moderate decrease upon mtSSB knockdown in HeLa cells. These results confirmed the requirement of mtSSB for mtDNA replication. Many molecules of mammalian mtDNA hold a short third strand, so-called 7S DNA, whose regulation is poorly understood. In contrast to the gradual decrease of mtDNA copy number, 7S DNA was severely reduced upon mtSSB knockdown in HeLa cells. Further, 7S DNA synthesis was significantly affected by mtSSB knockdown in an oseteosarcoma cell line. These data together suggest that mtSSB plays an important role in the maintenance of 7S DNA alongside its role in mtDNA replication. In addition, live-cell staining of mtDNA did not imply alteration in the organisation of mitochondrial nucleoid protein-mtDNA complexes upon mtSSB knockdown in HeLa cells. This result suggests that the presence of 7S DNA is not crucial for the organisation of mitochondrial nucleoids. (c) 2010 Elsevier B.V. All rights reserved.