Vinogradova M N, Gromova E S, Purmal' A A, Kosykh V G, Shabarova Z A
Mol Biol (Mosk). 1986 Sep-Oct;20(5):1329-36.
As shown by a nitrocellulose filter binding assay, in the absence of Mg2+ EcoRII restriction endonuclease binds specifically to a set of synthetic concatemer DNA duplexes of varying chain length, containing natural and modified recognition sites of this enzyme. The binding of the substrates with the central AT, TT or AA-pair in the recognition site decreases at AT greater than TT much greater than AA. Substitution of the pyrophosphate bond at the cleavage site for the phosphodiester or phosphoramide bond produces little influence on the stability of the complexes. The affinity of the enzyme for nonspecific sites is two orders of magnitude less than that for the specific EcoRII sequences. Equilibrium association constant for a substrate with one recognition site is 3.9 X 10(8) M-1. Addition of Mg2+ leads to the destabilization of the EcoRII endonuclease complex with DNA duplex, containing pyrophosphate bonds. The dissociation rate constants and the lifetime of the EcoRII endonuclease--synthetic substrates complexes have been determined.
如硝酸纤维素滤膜结合试验所示，在没有Mg2+的情况下，EcoRII限制性内切酶能特异性结合一组链长不同的合成串联DNA双链体，这些双链体包含该酶的天然和修饰识别位点。识别位点中带有中央AT、TT或AA碱基对的底物结合能力在AT时下降，下降程度为AT大于TT且远大于AA。将切割位点的焦磷酸键替换为磷酸二酯键或磷酰胺键对复合物的稳定性影响很小。该酶对非特异性位点的亲和力比对特异性EcoRII序列的亲和力低两个数量级。含有一个识别位点的底物的平衡缔合常数为3.9×10(8) M-1。添加Mg2+会导致EcoRII内切酶与含有焦磷酸键的DNA双链体复合物不稳定。已经测定了EcoRII内切酶与合成底物复合物的解离速率常数和寿命。
