Mitochondrial transcription terminator family members mTTF and mTerf5 have opposing roles in coordination of mtDNA synthesis.
Mitochondrial transcription terminator family members mTTF and mTerf5 have opposing roles in coordination of mtDNA synthesis.
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DOI：
10.1371/journal.pgen.1003800
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Jacobs HT
中科院分区：
文献类型：
--
作者：
Jõers P;Lewis SC;Fukuoh A;Parhiala M;Ellilä S;Holt IJ;Jacobs HT
所有基因组都需要一个系统来避免或处理DNA复制和转录机制之间的冲突。我们研究了果蝇中线粒体转录终止因子（mitochondrial transcription termination factor，mTERF）家族成员mTTF和mTerf 5在这一过程中的作用。果蝇mtDNA中的两个mTTF结合位点，也结合mTerf 5，被发现与复制暂停的主要位点一致。RNAi介导的基因敲除导致mtDNA耗竭和发育停滞。mTTF敲低降低了位点特异性复制暂停，但导致每个mTTF结合位点周围广泛区域的复制停滞和分叉退化增加。滞后链DNA合成受损，与延长RNA/DNA杂交片段复制中间体。这是伴随着重组中间体和有切口/断裂的mtDNA种类的积累。相反，mTerf 5敲低导致在mTTF结合位点的复制暂停增强，含有单链片段的脆弱复制中间体减少，以及含有RNA/DNA杂交片段的物种消失。这些发现表明，mTERF家族的蛋白质在转录和DNA复制的整合中具有重要的和以前未描述的作用，防止不受调节的碰撞，并促进两种机制之间的有效相互作用，这两种机制被推断为完成滞后链DNA合成所必需的。所有基因组都需要一个系统来防止DNA复制和转录机制之间的碰撞。我们研究了果蝇线粒体转录终止因子（mTERF）家族的两种蛋白在这一过程中的作用。这些因子，mTTF和mTerf 5，在线粒体基因组中共享共同的结合位点，我们发现这与复制暂停的位点一致。通过RNA干扰敲低任一因子导致mtDNA耗竭和发育停滞。mTTF敲低减少了位点特异性复制暂停，但导致随机停滞和复制叉的退化增加，滞后链的合成受损。我们把这归因于与转录机制的随机碰撞。相反，mTerf 5敲低导致在mTTF结合位点的复制暂停增强。这些发现表明，mTERF家族的蛋白质在转录和DNA复制的整合中具有重要的和以前未描述的作用，防止不受调节的碰撞，并促进两种机制之间的有效相互作用，这两种机制被推断为完成滞后链DNA合成所必需的。
All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis. All genomes require a system for preventing collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of two proteins of the mTERF (mitochondrial transcription termination factor) family in Drosophila. These factors, mTTF and mTerf5, share common binding sites in the mitochondrial genome, which we found to coincide with sites of replication pausing. Knockdown of either factor by RNA interference resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in random stalling and regression of replication forks, with impaired synthesis of the lagging strand. This we attribute to random collisions with the transcriptional machinery. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.
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