Crisona N J, Kanaar R, Gonzalez T N, Zechiedrich E L, Klippel A, Cozzarelli N R
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Mol Biol. 1994 Oct 28;243(3):437-57. doi: 10.1006/jmbi.1994.1671.
The Gin recombinase of phage Mu selectively mediates DNA inversion between two inversely oriented recombination sites (gix) and requires the assistance of three accessory factors: negative supercoiling, an enhancer sequence, and the protein Fis. Deletion and fusion reactions are proscribed. Recombination by Gin is selective because it occurs only through a particular synaptic complex tailored for inversion. A single amino acid change in Gin allows it to carry out deletion and fusion as well as inversion and to dispense with the requirement for the accessory factors. We investigated the recombination mechanism of a mutant Gin protein by analyzing the knotted products of processive recombination by electron microscopy and gel electrophoresis. We find that, in sharp contrast to wild-type Gin, mutant Gin recombines through a broad spectrum of synaptic complexes that differ topologically. We propose a model for the selectivity of wild-type Gin recombination that explains how the dependence on the accessory factors limits recombination to inversion. In addition, we show that processive recombination by wild-type Gin is not restricted by the number of base-pairs separating the gix sites from each other and from the enhancer. This result can be explained if strand exchange proceeds through alternative paths dictated by the energetics of DNA coiling.
噬菌体Mu的Gin重组酶可在两个反向排列的重组位点（gix）之间选择性地介导DNA倒位，且需要三种辅助因子的协助：负超螺旋、增强子序列和蛋白质Fis。禁止发生缺失和融合反应。Gin介导的重组具有选择性，因为它仅通过为倒位量身定制的特定突触复合体发生。Gin中的单个氨基酸变化使其能够进行缺失、融合以及倒位，并无需辅助因子。我们通过电子显微镜和凝胶电泳分析连续重组的纽结产物，研究了突变型Gin蛋白的重组机制。我们发现，与野生型Gin形成鲜明对比的是，突变型Gin通过多种拓扑结构不同的突触复合体进行重组。我们提出了一个关于野生型Gin重组选择性的模型，该模型解释了对辅助因子的依赖性如何将重组限制为倒位。此外，我们表明野生型Gin的连续重组不受gix位点之间以及与增强子之间碱基对数量的限制。如果链交换通过由DNA盘绕能量决定的替代路径进行，那么这一结果就可以得到解释。
