Stem-Loop Silencing Reveals that a Third Mitochondrial DNA Polymerase, POLID, Is Required for Kinetoplast DNA Replication in Trypanosomes
Stem-Loop Silencing Reveals that a Third Mitochondrial DNA Polymerase, POLID, Is Required for Kinetoplast DNA Replication in Trypanosomes
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DOI：
10.1128/ec.00199-08
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发表时间：
2008-12-01
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影响因子：
--
通讯作者：
Klingbeil, Michele M.
中科院分区：
文献类型：
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作者：
Chandler, Julian;Vandoros, Anthula V.;Klingbeil, Michele M.
动基体DNA（Kinetoplast DNA，kDNA）是锥虫的线粒体基因组，由数千个小环和数十个大环组成。拓扑结构的复杂性决定了一些不寻常的功能，包括一个拓扑异构酶介导的释放和再附着机制的小环复制和至少六个线粒体DNA聚合酶（Pos）的kDNA交易。在此之前，我们从布氏锥虫中鉴定出了四种与细菌DNA Pol I相似的A家族DNA Pol，并证明了其中两种（POLIB和POLIC）对于维持kDNA网络是必不可少的，而POLIA不是。在此，我们利用RNA干扰技术研究了POLID在原环T中的功能。布鲁塞。POLID的茎环沉默导致生长停滞和kDNA网络的逐渐丧失。kDNA复制中的其他缺陷包括小环和大环丰度的快速下降以及在kDNA网络丢失之前小环复制中间体的短暂积累。这些结果表明，POLID是kDNA复制所需的第三种必需DNA Pol。当其他真核生物利用单个DNA Pol（Pol gamma）复制线粒体DNA时，T.布鲁氏杆菌需要至少三个来维持复杂的kDNA网络。
Kinetoplast DNA ( kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol gamma) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network.