Ruben G, Spielman P, Tu C D, Jay E, Siegel B, Wu R
Nucleic Acids Res. 1977 Jun;4(6):1803-13. doi: 10.1093/nar/4.6.1803.
We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.
我们已经确定了在37摄氏度下，限制性内切酶EcoRI和HpaII对超螺旋SV40 DNA（I型）的切割模式。通过琼脂糖凝胶电泳分析和暗视野电子显微镜直接观察，我们发现，在线性SV40 DNA（III型）出现之前，会产生大量的单切口环状DNA（II型）。因此，两种限制性酶首先都只切割超螺旋DNA的一条链。互补链上的第二次切割在一段延迟期后发生。测定了EcoRI内切酶第二次切割的一级速率常数，并提出了两种酶的动力学反应方案。
