Alves J, Pingoud A, Haupt W, Langowski J, Peters F, Maass G, Wolff C
Eur J Biochem. 1984 Apr 2;140(1):83-92. doi: 10.1111/j.1432-1033.1984.tb08069.x.
We have investigated the influence of the nucleotide sequence adjacent to the recognition site on the rate of cleavage of DNA by the restriction endonuclease EcoRI. For this purpose two decadeoxynucleotides, d(G-G-G-A-A-T-T-C-T-T) (Ia) and d(A-A-G-A-A-T-T-C-C-C) (Ib) were synthesized. The duplex Ia X Ib is cleaved by EcoRI preferentially in the dA-rich strand (approximately 10 times over the dG-rich strand). The individual nucleotides Ia and Ib are also cleaved by EcoRI, Ib at a higher rate than Ia and both at a lower rate than Ia X Ib. The temperature dependence of the reaction rate shows that only double-stranded oligodeoxynucleotides are substrates for the EcoRI endonuclease. We have, furthermore, synthesized oligomers of d(G-G-A-A-T-T-C-C), which contain two, three and four EcoRI sites, respectively. These oligodeoxynucleotides are preferentially cleaved at the sites next to the 5' end, where the recognition site is only flanked by one dG X dC base pair, in contrast to the other sites which are flanked by three such pairs. These data indicate that sequences adjacent to the recognition site influence the rate of cleavage: dA X dT base pairs enhance and dG X dC base pairs slow down the hydrolytic activity of the EcoRI endonuclease.
我们研究了识别位点相邻的核苷酸序列对限制性内切酶EcoRI切割DNA速率的影响。为此合成了两条十聚脱氧核苷酸，d(G-G-G-A-A-T-T-C-T-T)（Ia）和d(A-A-G-A-A-T-T-C-C-C)（Ib）。双链Ia×Ib被EcoRI优先在富含dA的链上切割（比富含dG的链大约多10倍）。单个核苷酸Ia和Ib也能被EcoRI切割，Ib的切割速率高于Ia，两者的切割速率均低于Ia×Ib。反应速率的温度依赖性表明，只有双链寡聚脱氧核苷酸是EcoRI内切酶的底物。此外，我们合成了d(G-G-A-A-T-T-C-C)的寡聚物，它们分别含有两个、三个和四个EcoRI位点。这些寡聚脱氧核苷酸优先在5'端相邻的位点被切割，在这些位点识别位点仅由一个dG×dC碱基对侧翼，而其他位点由三个这样的碱基对侧翼。这些数据表明识别位点相邻的序列影响切割速率：dA×dT碱基对增强而dG×dC碱基对减缓EcoRI内切酶的水解活性。
