Duckett D R, Panis M J, Lilley D M
Department of Biochemistry The University, Dundee, U.K.
J Mol Biol. 1995 Feb 10;246(1):95-107. doi: 10.1006/jmbi.1994.0069.
Bacteriophage T7 endonuclease I is a resolving enzyme that selectively cleaves four-way DNA junctions, and related branched species. We have isolated mutants of this protein that retain full structural selectivity of binding to four-way junctions, but which are completely inactive as nucleases. This is consistent with a divisibility of structure-selective binding and catalysis. The mutations that inactivate endonuclease I as a nuclease are clustered into the second quarter of the primary sequence, a region that displays some sequence similarity with the related junction-resolving enzyme endonuclease VII from bacteriophage T4. This suggests that these residues may form the active site of these enzymes. The configuration of the helical arms of the junction bound by mutant endonuclease I has been investigated by gel electrophoretic methods. We find that the junction is bound in the presence or absence of magnesium ions, and that the global structure of the bound form is apparently identical with or without cations. The patterns of mobilities suggest that the structure of the junction becomes perturbed by the binding of the protein.
噬菌体T7核酸内切酶I是一种切割酶，可选择性切割四链DNA连接体及相关分支结构。我们已分离出该蛋白的突变体，这些突变体保留了与四链连接体结合的完整结构选择性，但作为核酸酶则完全无活性。这与结构选择性结合和催化的可分性是一致的。使核酸内切酶I失活的突变集中在一级序列的第二个四分之一区域，该区域与噬菌体T4的相关连接体切割酶核酸内切酶VII有一些序列相似性。这表明这些残基可能构成了这些酶的活性位点。已通过凝胶电泳方法研究了突变型核酸内切酶I所结合的连接体螺旋臂的构型。我们发现，无论有无镁离子，连接体均能被结合，且结合形式的整体结构在有或无阳离子的情况下显然相同。迁移率模式表明，连接体的结构因蛋白质的结合而受到干扰。
