Benjamin H W, Cozzarelli N R
Department of Molecular Biology, University of California, Berkeley 94720.
EMBO J. 1988 Jun;7(6):1897-905. doi: 10.1002/j.1460-2075.1988.tb03023.x.
We have isolated in quantitative yield the synaptic intermediate formed during site-specific recombination by Tn3 resolvase and characterized it by restriction endonuclease mapping, electron microscopy and topological methods. The intermediate accumulates at low reaction temperatures and is stabilized by crosslinking of the resolvase protomers with glutaraldehyde. The DNA-resolvase complex that maintains the structure of the intermediate (the synaptosome) is approximately 100 A in diameter, forms specifically at resolution (res) sites, and requires two res sites in a supercoiled DNA molecule. Resolvase bound to individual res sites protects approximately -0.5 supercoil per site from relaxation by a topoisomerase, whereas the formation of the synaptosome protects -3 supercoils and condenses the associated DNA to a supercoil density 2.5 times that of the non-complexed substrate. Although recombination requires two directly repeated res sites, both direct and inverted sites form synaptosomes. We conclude that the specificity of recombination is achieved by a three-stage recognition system: binding of resolvase to separate sites, formation of the synaptosome and determination of site orientation from within the complex.
我们已通过定量产率分离出由Tn3解离酶在位点特异性重组过程中形成的突触中间体，并通过限制性内切酶图谱分析、电子显微镜和拓扑学方法对其进行了表征。该中间体在低温反应条件下积累，并通过戊二醛使解离酶原聚体交联而得以稳定。维持中间体（突触体）结构的DNA-解离酶复合物直径约为100埃，特异性地在解离（res）位点形成，并且在超螺旋DNA分子中需要两个res位点。与单个res位点结合的解离酶可保护每个位点约-0.5个超螺旋不被拓扑异构酶松弛，而突触体的形成可保护-3个超螺旋，并将相关DNA浓缩至超螺旋密度为未复合底物的2.5倍。尽管重组需要两个直接重复的res位点，但正向和反向位点均可形成突触体。我们得出结论，重组的特异性是通过一个三阶段识别系统实现的：解离酶与分离位点的结合、突触体的形成以及从复合物内部确定位点方向。
