Janscak P, Abadjieva A, Firman K
Biophysics Laboratories School of Biological Sciences, University of Portsmouth, UK.
J Mol Biol. 1996 Apr 19;257(5):977-91. doi: 10.1006/jmbi.1996.0217.
In this paper we describe a two-plasmid system which allows over-production of the R.EcoR124I restriction endonuclease. The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification. Unexpectedly, the enzyme was found to require only ATP and Mg2+ for ATPase activity and DNA cleavage; S-adenosyl methionine (SAM), which has been described as a cofactor of type I restriction enzymes, is not required by R.EcoR124I. However, SAM was found to stimulate the rate of ATPase activity and DNA cleavage. This may occur through an increase in specific DNA binding by R.EcoR124I in the presence of SAM, as indicated by our surface plasmon resonance experiments. These functional differences from the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes, namely that the stoichiometry of R.EcoR124I appears to be R1M2S1 while that of R.EcoKI is R2M2S1. Supercoiled DNA with one or two SR124I recognition sites is cleaved by the same mechanism inferring co-operation between specifically bound and excess enzymes. Nicked-circle DNA is an intermediate of cleavage reaction. Cleavage of DNA was inhibited by an increased degree of negative supercoiling, which may reflect an increased difficulty for the enzyme to translocate the DNA. Hemi-methylated DNA was the preferred substrate for methylation.
在本文中，我们描述了一种双质粒系统，该系统可实现R.EcoR124I限制性内切酶的过量表达。该内切酶已被纯化至毫克级纯度，并且已证明其在限制和修饰方面均具有完全活性。出乎意料的是，发现该酶仅需ATP和Mg2+即可进行ATP酶活性和DNA切割；R.EcoR124I不需要已被描述为I型限制酶辅助因子的S-腺苷甲硫氨酸（SAM）。然而，发现SAM可刺激ATP酶活性和DNA切割的速率。如我们的表面等离子体共振实验所示，这可能是由于在SAM存在下R.EcoR124I与特定DNA的结合增加所致。与已充分描述的R.EcoKI限制性内切酶的这些功能差异反映在这两种酶之间可能存在的结构差异上，即R.EcoR124I的化学计量似乎是R1M2S1，而R.EcoKI的化学计量是R2M2S1。具有一个或两个SR124I识别位点的超螺旋DNA通过相同的机制被切割，这表明特异性结合的酶和过量的酶之间存在协同作用。带切口的环状DNA是切割反应的中间体。DNA切割受到负超螺旋程度增加的抑制，这可能反映了酶对DNA进行易位的难度增加。半甲基化DNA是甲基化的首选底物。
