在存在标准底物的情况下，限制内切酶R.EcoRII可切割识别位点中含有甲基化碱基或非核苷酸插入片段的DNA双链体。

DNA duplexes containing methylated bases or non-nucleotide inserts in the recognition site are cleaved by restriction endonuclease R.EcoRII in presence of canonical substrate.

作者信息

Petrauskene O V, Gromova E S, Romanova E A, Volkov E M, Oretskaya T S, Shabarova Z A

机构信息

Department of Chemistry, Moscow State University, Russia.

出版信息

Gene. 1995 May 19;157(1-2):173-6. doi: 10.1016/0378-1119(94)00737-d.

DOI:10.1016/0378-1119(94)00737-d

PMID:7607486

Abstract

DNA duplexes containing the natural methylated bases N6-methyladenine (m6Ade), N4-methylcytosine (m4Cyt) or C5-methylcytosine (m5Cyt) in one strand of the recognition sequence are resistant to EcoRII restriction endonuclease (R.EcoRII). Hydrolysis of these modified duplexes was observed in the presence of the canonical substrate. Incorporation of m4Cyt or m5Cyt into both strands of the recognition sequence precludes such activation by a canonical substrate. R.EcoRII also fails to cleave substrate analogs in which one of the nucleosides in the recognition site is replaced by the 1,2-dideoxyribose (D) or by 1,3-propanediol (Prd) (modeling DNA with an abasic site). The hydrolysis of DNA duplexes with non-nucleotide inserts is also activated in the presence of canonical substrate. Thus, the two-substrate mechanism of EcoRII-DNA interaction allows hydrolysis of apurinic/apyrimidinic and hemimethylated DNA.

摘要

在识别序列的一条链中含有天然甲基化碱基N6-甲基腺嘌呤（m6Ade）、N4-甲基胞嘧啶（m4Cyt）或C5-甲基胞嘧啶（m5Cyt）的DNA双链体对EcoRII限制性内切酶（R.EcoRII）具有抗性。在标准底物存在的情况下，观察到这些修饰双链体的水解。将m4Cyt或m5Cyt掺入识别序列的两条链中可阻止标准底物的这种激活作用。R.EcoRII也无法切割识别位点中的一个核苷被1,2-二脱氧核糖（D）或1,3-丙二醇（Prd）取代的底物类似物（模拟具有无碱基位点的DNA）。在标准底物存在的情况下，具有非核苷酸插入片段的DNA双链体的水解也会被激活。因此，EcoRII与DNA相互作用的双底物机制允许对无嘌呤/无嘧啶和半甲基化DNA进行水解。