Preferential relaxation of positively supercoiled DNA by E-coli topoisomerase IV in single-molecule and ensemble measurements
Preferential relaxation of positively supercoiled DNA by E-coli topoisomerase IV in single-molecule and ensemble measurements
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DOI：
10.1101/gad.838900
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发表时间：
2000-11-15
影响因子：
10.5
通讯作者：
Cozzarelli, NR
中科院分区：
文献类型：
--
作者：
Crisona, NJ;Strick, TR;Cozzarelli, NR
我们发现，正超螺旋[（+）SC] DNA是大肠杆菌拓扑异构酶IV（topo IV）的首选底物。我们测量拓扑IV松弛（-）和（+）超螺旋在真实的时间在单一的，拴系的DNA分子，以补充合奏实验。我们发现，（+）SC DNA的偏好是完全在低酶浓度。另外，topo IV以比（-）超螺旋快20倍的速率松弛（+）超螺旋，主要是由于（+）SC DNA的持续合成能力增加了约10倍。竞争实验中（+）SC DNA的优先切割表明，在存在或不存在ATP的情况下，底物歧视可以发生在链通过之前。我们提出拓扑IV通过识别（+）SC DNA的几何形状来区分（-）和（+）超螺旋DNA。我们的研究结果解释了topo IV如何快速去除（+）超螺旋以支持DNA复制，而不放松染色体的基本（-）超螺旋。他们还表明，topo IV的超螺旋松弛速率比迄今为止所认识到的要快几个数量级，因此在每个复制叉处，单个酶就足够了。
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.