Determination of the stereostructure of the product of Tn3 resolvase by a general method.
Determination of the stereostructure of the product of Tn3 resolvase by a general method.
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用通用方法测定 Tn3 解离酶产物的立体结构。
DOI：
10.1073/pnas.82.4.1079
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发表时间：
1985
影响因子：
11.1
通讯作者：
Cozzarelli,NR
中科院分区：
文献类型：
--
作者：
Wasserman,SA;Cozzarelli,NR
建立了一种测定DNA链烷绝对结构的方法。链式DNA在用RecA蛋白包被增稠并展开用于电子显微镜之前部分变性。这种处理允许可视化每个环的方向以及在交叉点处识别上覆和下伏DNA。这些测定定义了链烷的拓扑结构，为测试DNA重组和复制中链烷产生酶的机制提供了强有力的手段。该技术被用来显示由Tn 3解离酶介导的位点特异性重组的连锁产物的单互锁是唯一的负符号。独特的拓扑结构的产品表明，resolvase固定在突触的重组位点之间的超螺旋的数量和这样的超螺旋的数量在链交换过程中丢失或获得的总和。这些数据有力地表明，事实上有三个负超螺旋之间的突触网站;一个超螺旋是溶解的交叉机制，而其他两个变态成独特的索烃联锁。
A method has been developed for determination of the absolute structure of DNA catenanes. The catenated DNA is partially denatured before being thickened with a coating of RecA protein and spread for electron microscopy. This treatment allows visualization of the orientation of each ring as well as identification of the overlying and underlying DNAs at crossing points. These determinations define the topology of a catenane, providing a powerful means for testing mechanisms of catenane-producing enzymes in DNA recombination and replication. The technique was used to show that the single interlock of the catenated products of site-specific recombination mediated by Tn3 resolvase is exclusively of negative sign. The unique topology of the products indicates that resolvase fixes the sum of the number of supercoils between recombination sites at synapsis and the number of such supercoils lost or gained during strand exchange. The data strongly suggest that there are in fact three negative supercoils between synapsed sites; one supercoil is dissolved in the cross-over mechanism, whereas the other two are metamorphosed into the unique catenane interlock.