Roles of topoisomerase IV and DNA gyrase in DNA unlinking during replication in Escherichia coli.
Roles of topoisomerase IV and DNA gyrase in DNA unlinking during replication in Escherichia coli.
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DOI：
10.1101/gad.9.22.2859
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发表时间：
1995-11
影响因子：
10.5
通讯作者：
E. Zechiedrich;N. Cozzarelli
中科院分区：
文献类型：
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作者：
E. Zechiedrich;N. Cozzarelli
为了让细胞完成DNA复制，必须通过拓扑异构酶去除Watson-Crick链之间的每一条连接。在此之前，我们曾报道过拓扑异构酶IV(Topo IV)的抑制会导致连接的质粒复制子积累到大约10%的稳定水平。在大肠杆菌中用[~3H]胸腺嘧啶核苷脉冲标记，我们发现在没有Topo IV活性的情况下，几乎所有新合成的质粒DNA都是连锁的。脉冲追踪实验表明，即使在没有Topo IV的情况下，链烷也会被代谢，剩余的周转是由DNA旋转酶以大约0.01/秒的速度进行的。利用极短的脉冲标记时间，我们在野生型细胞中发现了大量的复制链烷。在野生型细胞中，Topo IV和DNA旋转酶的联合活性导致的连环芳烃解链速率约为1/秒。因此，在体内，旋转酶的效率比Topo IV低100倍。这可能解释了为什么一个功能齐全的旋转酶不能阻止新合成的质粒DNA的连锁反应和Topo IV突变体的分配表型。我们的结论是，链烷是DNA复制的动力学中间体，Topo IV的基本作用是解链子体复制子。
For a cell to complete DNA replication, every link between the Watson-Crick strands must be removed by topoisomerases. Previously, we reported that the inhibition of topoisomerase IV (topo IV) leads to the accumulation of catenated plasmid replicons to a steady-state level of approximately 10%. Using pulse labeling with [3H]thymidine in Escherichia coli, we have found that in the absence of topo IV activity, nearly all newly synthesized plasmid DNA is catenated. Pulse-chase protocols revealed that catenanes are metabolized even in the absence of topo IV and that the residual turnover is carried out by DNA gyrase at a rate of approximately 0.01/sec. Using extremely short pulse-labeling times, we identified significant amounts of replication catenanes in wild-type cells. The rate of catenane unlinking in wild-type cells by the combined activities of topo IV and DNA gyrase was approximately 1/sec. Therefore, gyrase is 100-fold less efficient than topo IV in plasmid replicon decatenation in vivo. This may explain why a fully functional gyrase cannot prevent the catenation of newly synthesized plasmid DNA and the partition phenotype of topo IV mutants. We conclude that catenanes are kinetic intermediates in DNA replication and that the essential role of topo IV is to unlink daughter replicons.