Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA.
Mol Microbiol. 2010 Mar;75(6):1414-25. doi: 10.1111/j.1365-2958.2010.07061.x. Epub 2009 Feb 1.
The unique mitochondrial DNA of trypanosomes is a catenated network of minicircles and maxicircles called kinetoplast DNA (kDNA). The network is essential for survival, and requires an elaborate topoisomerase-mediated release and reattachment mechanism for minicircle theta structure replication. At least seven DNA polymerases (pols) are involved in kDNA transactions, including three essential proteins related to bacterial DNA pol I (POLIB, POLIC and POLID). How Trypanosoma brucei utilizes multiple DNA pols to complete the topologically complex task of kDNA replication is unknown. To fill this gap in knowledge we investigated the cellular role of POLIB using RNA interference (RNAi). POLIB silencing resulted in growth inhibition and progressive loss of kDNA networks. Additionally, unreplicated covalently closed precursors become the most abundant minicircle replication intermediate as minicircle copy number declines. Leading and lagging strand minicircle progeny similarly declined during POLIB silencing, indicating POLIB had no apparent strand preference. Interestingly, POLIB RNAi led to the accumulation of a novel population of free minicircles that is composed mainly of covalently closed minicircle dimers. Based on these data, we propose that POLIB performs an essential role at the core of the minicircle replication machinery.
锥虫独特的线粒体 DNA 是一种被称为动基体 DNA（kDNA）的微小环和大环的链状网络。该网络对生存至关重要，需要一种精细的拓扑异构酶介导的释放和重新附着机制，以实现微小环θ结构的复制。至少有七种 DNA 聚合酶（pols）参与 kDNA 交易，包括与细菌 DNA pol I（POLIB、POLIC 和 POLID）相关的三种必需蛋白。布氏锥虫如何利用多种 DNA pol 完成 kDNA 复制这种拓扑复杂的任务尚不清楚。为了填补这一知识空白，我们使用 RNA 干扰（RNAi）研究了 POLIB 的细胞作用。POLIB 沉默导致生长抑制和 kDNA 网络的逐渐丧失。此外，由于微小环拷贝数下降，未复制的共价闭合前体成为最丰富的微小环复制中间体。在 POLIB 沉默期间，前导链和滞后链微小环后代也同样减少，表明 POLIB 没有明显的链偏好。有趣的是，POLIB RNAi 导致一种新型游离微小环的积累，该微小环主要由共价闭合的微小环二聚体组成。基于这些数据，我们提出 POLIB 在微小环复制机制的核心中发挥着重要作用。
