Kubareva E A, Gromova E S, Pein C D, Krug A, Oretskaya T S, Cech D, Shabarova Z A
A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, U.S.S.R.
Biochim Biophys Acta. 1991 Mar 26;1088(3):395-400. doi: 10.1016/0167-4781(91)90131-5.
To elucidate the mechanism of action of the restriction endonucleases--isoschizomers EcoRII and MvaI--a study was made of their interaction with a set of synthetic oligonucleotide duplexes containing a single 5'-d(CCA/TGG)-3' EcoRII (MvaI) recognition site. The substrates had varying length and structure of the nucleotide sequences flanking the recognition site. The structure of the flanking sequence is important for the cleavage by EcoRII and MvaI enzymes; there is a structure which was found to speed up the EcoRII and MvaI action. The cleavage of oligonucleotide duplexes by EcoRII enzyme does not go to completion. EcoRII endonuclease cleaved extended substrates less efficiently than short ones. Extension of the flanking sequences, with the same nucleotide surrounding of the recognition site, substantially altered the whole kinetic pattern of MvaI hydrolysis. This was not observed with EcoRII enzyme. The restriction endonuclease MvaI distinguished between dA and dT residues in the recognition site, which was reflected in the higher rate of hydrolysis of the dA-containing strand of the quasi-palindromic DNA duplex.
为阐明限制性核酸内切酶同裂酶EcoRII和MvaI的作用机制，对它们与一组含有单个5'-d(CCA/TGG)-3'EcoRII（MvaI）识别位点的合成寡核苷酸双链体的相互作用进行了研究。底物在识别位点两侧的核苷酸序列长度和结构各不相同。侧翼序列的结构对于EcoRII和MvaI酶的切割很重要；发现有一种结构能加速EcoRII和MvaI的作用。EcoRII酶对寡核苷酸双链体的切割不完全。EcoRII核酸内切酶切割延伸底物的效率低于短底物。在识别位点周围核苷酸相同的情况下，侧翼序列的延伸显著改变了MvaI水解的整个动力学模式。而EcoRII酶未观察到这种情况。限制性核酸内切酶MvaI能区分识别位点中的dA和dT残基，这反映在准回文DNA双链体中含dA链的水解速率较高。
