Peng H, Marians K J
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8571-5. doi: 10.1073/pnas.90.18.8571.
Topoisomerase IV (Topo IV), encoded by parC and parE, is required for partition of the daughter chromosomes in Escherichia coli. This enzyme is likely responsible for decatenating the linked daughter chromosomes after replication. In this report, we have examined the action of Topo IV in both pBR322 and oriC DNA replication reconstituted in vitro with purified proteins. Gyrase fails to decatenate the linked daughter molecules under any condition in the oriC system and at physiological salt concentrations in the pBR322 system, whereas Topo IV stimulates generation of monomer product DNA by 7- to 10-fold. Topo IV-catalyzed decatenation of isolated multiply linked DNA dimers was relatively insensitive to salt; it proceeded at 14% of the maximal rate even in the presence of 800 mM potassium glutamate. In contrast, decatenation in vitro by gyrase was inhibited completely under these conditions. Pulse-chase analysis indicated that Topo IV-catalyzed resolution of linked daughter DNA molecules occurred prior to completion of DNA replication, such that multiply linked daughter molecules did not arise. These results suggest that during DNA replication, gyrase acts primarily to relieve accumulated positive supercoiling and Topo IV acts to segregate the daughter chromosomes.
由parC和parE编码的拓扑异构酶IV（Topo IV）是大肠杆菌中子代染色体分离所必需的。这种酶可能负责在复制后解开相连的子代染色体。在本报告中，我们研究了Topo IV在体外利用纯化蛋白重建的pBR322和oriC DNA复制中的作用。在oriC系统的任何条件下以及在pBR322系统的生理盐浓度下，回旋酶都无法解开相连的子代分子，而Topo IV可将单体产物DNA的生成刺激7至10倍。Topo IV催化的分离的多重连接DNA二聚体的解连环对盐相对不敏感；即使在存在800 mM谷氨酸钾的情况下，它也以最大速率的14%进行。相比之下，在这些条件下，回旋酶的体外解连环作用被完全抑制。脉冲追踪分析表明，Topo IV催化的相连子代DNA分子的解离发生在DNA复制完成之前，因此不会出现多重连接的子代分子。这些结果表明，在DNA复制过程中，回旋酶主要作用是缓解积累的正超螺旋，而Topo IV作用是分离子代染色体。
