Miller Jonathan C, Delzell Stephanie B, Concepción-Acevedo Jeniffer, Boucher Michael J, Klingbeil Michele M
Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA.
Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, 1600 Clifton Road, Atlanta, GA 30329, USA.
J Cell Sci. 2020 May 7;133(9):jcs233072. doi: 10.1242/jcs.233072.
Mitochondrial DNA of and related parasites is a catenated network containing thousands of minicircles and tens of maxicircles, called kinetoplast DNA (kDNA). Replication of a single nucleoid requires at least three DNA polymerase I-like proteins (i.e. POLIB, POLIC and POLID), each showing discrete localizations near the kDNA during S phase. POLIB and POLID have roles in minicircle replication but the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNA interference (RNAi)-complementation system to dissect the functions of two distinct POLIC regions, i.e. the conserved family A DNA polymerase (POLA) domain and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization of kDNA, active site point mutations in the POLA domain impaired minicircle replication similar to that of POLIB and POLID depletions. Complementation with POLA domain alone abolished the formation of POLIC foci and partially rescued the RNAi phenotype. Furthermore, we provide evidence that the UCR is crucial in cell cycle-dependent protein localization and facilitates proper distribution of progeny networks. This is the first report of a DNA polymerase that impacts on mitochondrial nucleoid distributionThis article has an associated First Person interview with the first author of the paper.
锥虫及相关寄生虫的线粒体DNA是一个包含数千个微环和数十个大环的连环网络，称为动基体DNA（kDNA）。单个核仁的复制至少需要三种DNA聚合酶I样蛋白（即POLIB、POLIC和POLID），在S期，每种蛋白在kDNA附近均表现出离散定位。POLIB和POLID在微环复制中发挥作用，但POLIC在kDNA维持中的具体作用尚不清楚。在这里，我们使用RNA干扰（RNAi）互补系统来剖析两个不同的POLIC区域的功能，即保守的A家族DNA聚合酶（POLA）结构域和未表征的N端区域（UCR）。虽然用野生型POLIC进行RNAi互补可恢复kDNA含量和kDNA的细胞周期定位，但POLA结构域中的活性位点点突变会损害微环复制，类似于POLIB和POLID缺失的情况。仅用POLA结构域进行互补会消除POLIC焦点的形成，并部分挽救RNAi表型。此外，我们提供的证据表明，UCR在细胞周期依赖性蛋白定位中至关重要，并有助于子代网络的正确分布。这是关于一种影响线粒体核仁分布的DNA聚合酶的首次报道。本文配有对该论文第一作者的第一人称访谈。
