Crisona N J, Strick T R, Bensimon D, Croquette V, Cozzarelli N R
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Genes Dev. 2000 Nov 15;14(22):2881-92. doi: 10.1101/gad.838900.
We show that positively supercoiled [(+) SC] DNA is the preferred substrate for Escherichia coli topoisomerase IV (topo IV). We measured topo IV relaxation of (-) and (+) supercoils in real time on single, tethered DNA molecules to complement ensemble experiments. We find that the preference for (+) SC DNA is complete at low enzyme concentration. Otherwise, topo IV relaxed (+) supercoils at a 20-fold faster rate than (-) supercoils, due primarily to about a 10-fold increase in processivity with (+) SC DNA. The preferential cleavage of (+) SC DNA in a competition experiment showed that substrate discrimination can take place prior to strand passage in the presence or absence of ATP. We propose that topo IV discriminates between (-) and (+) supercoiled DNA by recognition of the geometry of (+) SC DNA. Our results explain how topo IV can rapidly remove (+) supercoils to support DNA replication without relaxing the essential (-) supercoils of the chromosome. They also show that the rate of supercoil relaxation by topo IV is several orders of magnitude faster than hitherto appreciated, so that a single enzyme may suffice at each replication fork.
我们发现，正超螺旋[（+）SC] DNA是大肠杆菌拓扑异构酶IV（拓扑异构酶IV）的首选底物。我们在单个拴系的DNA分子上实时测量了拓扑异构酶IV对（-）和（+）超螺旋的松弛作用，以补充整体实验。我们发现，在低酶浓度下，对（+）SC DNA的偏好是完全的。否则，拓扑异构酶IV松弛（+）超螺旋的速度比（-）超螺旋快20倍，这主要是因为与（+）SC DNA结合时，持续合成能力增加了约10倍。在竞争实验中，（+）SC DNA的优先切割表明，在有或没有ATP的情况下，底物识别可以在链通过之前发生。我们提出，拓扑异构酶IV通过识别（+）SC DNA的几何结构来区分（-）和（+）超螺旋DNA。我们的结果解释了拓扑异构酶IV如何能够快速去除（+）超螺旋以支持DNA复制，而不松弛染色体中必不可少的（-）超螺旋。它们还表明，拓扑异构酶IV松弛超螺旋的速度比迄今所认识到的快几个数量级，因此每个复制叉处可能只需一种酶就足够了。
