From the Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
From the Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
J Biol Chem. 2014 Jun 27;289(26):18595-602. doi: 10.1074/jbc.M114.555367. Epub 2014 May 5.
Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin OH, in the ribosomal genes (12S and 16S) and at the light strand replication origin OL. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.
线粒体拓扑异构酶 I（Top1mt）是一种存在于脊椎动物中的 I 型 B 拓扑异构酶，专门靶向线粒体。Top1mt 通过引入瞬时断裂复合物来松弛线粒体 DNA（mtDNA）超螺旋，其中断裂的 DNA 链围绕完整的链旋转。Top1mt 断裂复合物（Top1mtcc）可以在体外通过喜树碱（CPT）稳定。然而，CPT 不能有效地在细胞中捕获 Top1mtcc，并且由于核 Top1 靶向而具有高细胞毒性。为了在体内将 Top1mtcc 映射到 mtDNA 上并克服 CPT 的限制，我们设计了 Top1mt 中的两个取代（T546A 和 N550H）来稳定 Top1mtcc。我们将双突变酶称为 Top1mt*。使用 Top1mt的逆转录病毒转导和 ChIP-on-chip 分析在 Top1mt 敲除的鼠胚胎成纤维细胞中，我们证明 Top1mt优先在 mtDNA 的非编码调节区域形成高水平的断裂复合物，特别是在重链复制起始 OH 处、核糖体基因（12S 和 16S）和轻链复制起始 OL 处积累。Top1mt*的表达也会导致 mtDNA 迅速耗竭而不影响线粒体质量，这表明存在特定的线粒体途径来去除受损的 mtDNA。
