Contributions of supercoiling to Tn3 resolvase and phage Mu Gin site-specific recombination
Contributions of supercoiling to Tn3 resolvase and phage Mu Gin site-specific recombination
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DOI：
10.1006/jmbi.1996.0067
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发表时间：
1996-02-16
影响因子：
5.6
通讯作者：
Cozzarelli, NR
中科院分区：
文献类型：
--
作者：
Benjamin, KR;Abola, AP;Cozzarelli, NR
位点特异性重组酶的解离酶/转化酶家族的成员需要含有两个重组位点的超螺旋底物。为了剖析超螺旋在Tn 3和γ δ分解酶和噬菌体Mu Gin转化酶重组中的作用，我们使用了提供标准底物的一些但不是全部拓扑特征的底物。我们将Tn 3解离酶反应分为突触和突触后两个阶段。我们发现超螺旋对每个阶段的贡献是不同的，因为在没有超螺旋或低水平的底物超螺旋的情况下，底物连锁足以用于突触，但不足以用于突触后。利用结构和功能拓扑分析，我们证实了解离酶突触复合物与切口索烃重组中间体。对超螺旋的要求甚至不太严格的γ δ解离酶，它重组切口索烃约一半，因为它没有超螺旋基板。即使重组增强子位于切口环上，也会发生索烃的Gin重组，只要两个交换位点都位于超螺旋环上。因此，在Gin交叉位点需要超螺旋化，而在增强子不需要。我们的结论是，只有构象效应的超螺旋所需的resolvase突触和鳃增强子的功能，但扭转效应，可能是双螺旋解旋，需要Tn 3 resolvase突触后和在Gin重组位点。(C)出版社：Academic Press Limited。
Members of the resolvase/invertase family of site-specific recombinases require supercoiled substrates containing two recombination sites. To dissect the roles of supercoiling in recombination by the Tn3 and gamma delta resolvases and the phage Mu Gin invertase, we used substrates that provided some but not all of the topological features of the standard substrate. We divided the Tn3 resolvase reaction into two stages, synapsis and postsynapsis. We found the contributions of supercoiling to each stage were distinct, since substrate catenation in the absence of supercoiling or low levels of substrate supercoiling were sufficient for synapsis but not postsynapsis. Using structural and functional topological analyses, we verified that the resolvase synaptic complexes with nicked catenanes were recombination intermediates. The requirement for supercoiling was even less stringent for the gamma delta resolvase, which recombined nicked catenanes about half as well as it did supercoiled substrates. Gin recombination of catenanes occurred even if the recombinational enhancer was on a nicked ring, as long as both crossover sites were on a supercoiled ring. Therefore, supercoiling is required at the Gin crossover sites but not at the enhancer. We conclude that solely conformational effects of supercoiling are required for resolvase synapsis and the function of the Gill enhancer, but that a torsional effect, probably double helix unwinding, is needed for Tn3 resolvase postsynapsis and at the Gin recombination sites. (C) 1996 Academic Press Limited.