Decatenating activity of Escherichia coli DNA gyrase and topoisomerases I and III during oriC and pBR322 DNA replication in vitro.
Decatenating activity of Escherichia coli DNA gyrase and topoisomerases I and III during oriC and pBR322 DNA replication in vitro.
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DOI：
10.1016/s0021-9258(17)42140-5
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发表时间：
1994-01
期刊：
影响因子：
--
通讯作者：
H. Hiasa;R. DiGate;K. Marians
中科院分区：
文献类型：
--
作者：
H. Hiasa;R. DiGate;K. Marians
用纯化的复制蛋白重建的oriC和pBR322 DNA复制已被用于研究大肠杆菌拓扑异构酶I、DNA促旋酶和拓扑异构酶III在DNA复制的最后阶段的功能活性。在oriC系统中，DNA回旋酶催化的子DNA分子的脱连环是非常低效的，而拓扑异构酶III可以催化完全脱连环。在pBR 322 DNA复制系统中，几乎所有的子DNA分子都能在无盐条件下被DNA旋转酶脱链。通过加入生理浓度的盐，在pBR 322系统中DNA促旋酶的脱连环被完全抑制，而不伴随DNA合成的抑制。然而，拓扑异构酶III可以使pBR 322系统中的所有子DNA分子解链，即使在高浓度盐的存在下。在oriC系统中没有观察到类似的效果，因为盐的加入抑制了DNA的合成。拓扑异构酶I不能在oriC或pBR322复制系统中检测的任何条件下催化去连环化。在复制系统中加入拓扑异构酶I只会抑制DNA的合成。
oriC and pBR322 DNA replication, reconstituted with purified replication proteins, has been used to study the functional activities of Escherichia coli topoisomerase I, DNA gyrase, and topoisomerase III during the final stages of DNA replication. In the oriC system, DNA gyrase-catalyzed decatenation of daughter DNA molecules was very inefficient, whereas topoisomerase III could catalyze complete decatenation. In the pBR322 DNA replication system, almost all the daughter DNA molecules could be decatenated by DNA gyrase alone in the absence of salt. Decatenation by DNA gyrase in the pBR322 system was completely inhibited, without a concomitant inhibition of DNA synthesis, by the addition of physiological concentrations of salt. Topoisomerase III, however, could decatenate all of the daughter DNA molecules in the pBR322 system, even in the presence of high concentrations of salt. A similar effect could not be observed in the oriC system, because the addition of salt inhibited DNA synthesis. Topoisomerase I was incapable of catalyzing decatenation under any conditions examined in either the oriC or pBR322 replication system. The addition of topoisomerase I to the replication systems resulted only in an inhibition of DNA synthesis.