Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
PLoS One. 2018 Aug 17;13(8):e0202057. doi: 10.1371/journal.pone.0202057. eCollection 2018.
Restriction enzymes are the bread and butter of Molecular Biology. Nonetheless, how restriction enzymes recognize and cleave their target is not always clear. When developing a method for the enzymatic production of oligonucleotides, we noticed that type II endonucleases BtsCI and BseGI, which recognize the sequence GGATGNN^, perform incomplete digestions of DNA hairpins, with the top strand nick not always occurring correctly. We tested the cutting of synthetic hairpins containing all possible combinations of dinucleotides following the recognition site and our results show that all sequences containing one adenine following GGATG were digested more efficiently. We further show that the same sequence preference is also observable in double stranded DNA at higher Mg2+ concentrations and even in optimal conditions. Kinetic results show that BtsCI has a noteworthy difference in the first-rate constants between different sequences and between the two catalytic domains. An increase in Mg2+ resulted in a drastic decrease in the catalytic activity of the top (sense) strand that wasn't always accompanied by a nick in the bottom strand (antisense).
限制酶是分子生物学的基石。然而，限制酶如何识别和切割其靶标并不总是清楚的。在开发酶促生产寡核苷酸的方法时，我们注意到识别序列 GGATGNN^的 II 型内切酶 BtsCI 和 BseGI 对 DNA 发夹进行不完全消化，顶部链的切口并不总是正确发生。我们测试了含有识别位点后所有可能的二核苷酸组合的合成发夹的切割，我们的结果表明，所有含有 GGATG 后一个腺嘌呤的序列都被更有效地消化。我们进一步表明，在更高的 Mg2+浓度下，双链 DNA 甚至在最佳条件下也可以观察到相同的序列偏好。动力学结果表明，BtsCI 在不同序列和两个催化结构域之间的第一速率常数上有显著差异。增加 Mg2+会导致顶部（有意义）链的催化活性急剧下降，而底部（反义）链的切口并不总是伴随着。
