Pöhler J R, Giraud-Panis M J, Lilley D M
CRC Nucleic Acid Structure Research Group, Department of Biochemistry, University, Dundee, UK.
J Mol Biol. 1996 Aug 2;260(5):678-96. doi: 10.1006/jmbi.1996.0430.
Bacteriophage T4 endonuclease VII is a nuclease that is selective for four-way DNA junctions and related branched DNA species. Using site-directed mutagenesis we have isolated a mutant protein (E86A) that is inactive in the cleavage of DNA junctions while retaining full selectivity of binding. Using endonuclease VII E86A we have shown: (1) The protein binds as a dimer to DNA junctions, with rapid exchange of subunits in free solution. (2) Binding to junctions is highly selective for the structure of DNA junctions; the complex is not displaced by a 1000-fold excess of duplex competitor DNA. (3) On binding endonuclease VII E86A to junctions, the configuration of the helical arms is significantly altered to a structure that is independent of the presence or absence of metal ions. We suggest a model for the structure of the junction in the protein complex. (4) The protein can bind to the junction in two stereochemically equivalent ways, depending upon the sequence of the junction. T4 endonuclease VII is a junction-selective enzyme that both recognises and manipulates the structure of its substrate.
噬菌体T4核酸内切酶VII是一种对四链DNA连接点及相关分支DNA种类具有选择性的核酸酶。我们通过定点诱变分离出一种突变蛋白（E86A），它在切割DNA连接点时无活性，但保留了完全的结合选择性。利用核酸内切酶VII E86A，我们已经证明：（1）该蛋白以二聚体形式与DNA连接点结合，在游离溶液中亚基快速交换。（2）与连接点的结合对DNA连接点的结构具有高度选择性；该复合物不会被1000倍过量的双链竞争DNA取代。（3）核酸内切酶VII E86A与连接点结合时，螺旋臂的构型会显著改变为一种与金属离子存在与否无关的结构。我们提出了蛋白质复合物中连接点结构的模型。（4）根据连接点的序列，该蛋白可以两种立体化学等效的方式与连接点结合。T4核酸内切酶VII是一种连接点选择性酶，它既能识别又能操纵其底物的结构。
