[EcoRII限制酶与修饰酶与合成DNA片段的相互作用。X. 具有结构异常的底物的水解作用]

[Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. X. Hydrolysis of substrates with structural abnormalities].

作者信息

Kubareva E A, Gromova E S, Oretskaia T S, Shabarova Z A

出版信息

Bioorg Khim. 1987 Sep;13(9):1205-11.

Abstract

Interaction of the EcoRII restriction endonuclease with a set of 30-membered substrates having structural anomalies in the recognition site (decreases CCT/AGG) and in adjacent sequences has been studied. A nick in the centre of the EcoRII recognition site between dC and dA residues slows down hydrolysis of the nonmodified strand, whereas the modified one is not cleaved. Removal of the phosphate group from the nick in this substrate does not alter the rate of the cleavage. The absence of one of the phosphate groups in the flanking sequence at a two-base-pair "distance" from the recognition site slows down the enzymatic hydrolysis. Removal of dA or dT out of the EcoRII recognition site blocks the enzymatic reaction. It appears that EcoRII does not interact with the phosphate group between dC and dA residues in the recognition site. Suggestions are made concerning possible contacts of the EcoRII restriction endonuclease with dA- and dT-residues of the recognition site and with the sugar-phosphate backbone of the adjacent nucleotide sequences.

摘要

研究了EcoRII限制性内切核酸酶与一组在识别位点（降低CCT/AGG）及相邻序列中存在结构异常的30元底物之间的相互作用。在EcoRII识别位点dC和dA残基之间的中心位置有一个切口，这会减缓未修饰链的水解，而修饰链则不会被切割。从该底物的切口中去除磷酸基团不会改变切割速率。在距识别位点两个碱基对“距离”的侧翼序列中缺少一个磷酸基团会减缓酶促水解。从EcoRII识别位点中去除dA或dT会阻断酶促反应。似乎EcoRII不会与识别位点中dC和dA残基之间的磷酸基团相互作用。针对EcoRII限制性内切核酸酶与识别位点的dA和dT残基以及相邻核苷酸序列的糖磷酸骨架之间可能的接触提出了相关建议。