Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2012 Oct 2;109(40):16125-30. doi: 10.1073/pnas.1206480109. Epub 2012 Sep 18.
Type IB topoisomerases (Top1Bs) relax excessive DNA supercoiling associated with replication and transcription by catalyzing a transient nick in one strand to permit controlled rotation of the DNA about the intact strand. The natural compound camptothecin (CPT) and the cancer chemotherapeutics derived from it, irinotecan and topotecan, are highly specific inhibitors of human nuclear Top1B (nTop1). Previous work on vaccinia Top1B led to an elegant model that describes a straightforward dependence of rotation and religation on the torque caused by supercoiling. Here, we used a single-molecule DNA supercoil relaxation assay to measure the torque dependence of nTop1 and its inhibition by CPT. For comparison, we also examined mitochondrial Top1B and an N-terminal deletion mutant of nTop1. Despite substantial sequence homology in their core domains, nTop1 and mitochondrial Top1B exhibit dramatic differences in sensitivity to torque and CPT, with the N-terminal deletion mutant of nTop1 showing intermediate characteristics. In particular, nTop1 displays nearly torque-independent religation probability, distinguishing it from other Top1B enzymes studied to date. Kinetic modeling reveals a hitherto unobserved torque-independent transition linking the DNA rotation and religation phases of the enzymatic cycle. The parameters of this transition determine the torque sensitivity of religation and the efficiency of CPT binding. This "kinetic clutch" mechanism explains the molecular basis of CPT sensitivity and more generally provides a framework with which to interpret Top1B activity and inhibition.
I 型拓扑异构酶（Top1Bs）通过催化一条链上的短暂切口，使 DNA 围绕完整链进行可控旋转，从而松弛与复制和转录相关的过度 DNA 超螺旋。天然化合物喜树碱（CPT）及其衍生的癌症化疗药物伊立替康和拓扑替康，是高度特异性的人核 Top1B（nTop1）抑制剂。以前对痘病毒 Top1B 的研究导致了一个优雅的模型，该模型描述了旋转和重连接与超螺旋引起的扭矩之间的直接关系。在这里，我们使用单分子 DNA 超螺旋松弛测定法来测量 nTop1 的扭矩依赖性及其对 CPT 的抑制作用。为了进行比较，我们还检查了线粒体 Top1B 和 nTop1 的 N 端缺失突变体。尽管它们的核心结构域具有高度的序列同源性，但 nTop1 和线粒体 Top1B 在对扭矩和 CPT 的敏感性方面表现出显著差异，nTop1 的 N 端缺失突变体表现出中间特征。特别是，nTop1 显示出几乎与扭矩无关的重连接概率，使其与迄今为止研究过的其他 Top1B 酶区分开来。动力学建模揭示了一个以前未观察到的与 DNA 旋转和重连接酶循环阶段相关的扭矩独立过渡。该过渡的参数决定了重连接的扭矩敏感性和 CPT 结合的效率。这种“动力学离合器”机制解释了 CPT 敏感性的分子基础，更普遍地为解释 Top1B 活性和抑制提供了一个框架。
