Laboratory of Biological DNA Modification, Institute of Biotechnology, Graiciūno 8, LT-02241 Vilnius, Lithuania.
Nucleic Acids Res. 2009 Nov;37(21):7332-41. doi: 10.1093/nar/gkp772.
DNA cytosine-5 methyltransferases (C5-MTases) are valuable models to study sequence-specific modification of DNA and are becoming increasingly important tools for biotechnology. Here we describe a structure-guided rational protein design combined with random mutagenesis and selection to change the specificity of the HhaI C5-MTase from GCGC to GCG. The specificity change was brought about by a five-residue deletion and introduction of two arginine residues within and nearby one of the target recognizing loops. DNA protection assays, bisulfite sequencing and enzyme kinetics showed that the best selected variant is comparable to wild-type M.HhaI in terms of sequence fidelity and methylation efficiency, and supersedes the parent enzyme in transalkylation of DNA using synthetic cofactor analogs. The designed C5-MTase can be used to produce hemimethylated CpG sites in DNA, which are valuable substrates for studies of mammalian maintenance MTases.
DNA 胞嘧啶-5 甲基转移酶（C5-MTases）是研究 DNA 序列特异性修饰的有价值模型，并且正在成为生物技术中越来越重要的工具。在这里，我们描述了一种结构指导的合理蛋白质设计，结合随机诱变和选择，将 HhaI C5-MTase 的特异性从 GCGC 改变为 GCG。特异性的改变是通过在靶识别环之一内和附近的五个残基缺失和引入两个精氨酸残基引起的。DNA 保护分析、亚硫酸氢盐测序和酶动力学表明，最佳选择的变体在序列保真度和甲基化效率方面与野生型 M.HhaI 相当，并且在使用合成辅因子类似物进行 DNA 转烷基化方面优于亲本酶。设计的 C5-MTase 可用于在 DNA 中产生半甲基化的 CpG 位点，这是研究哺乳动物维持 MTases 的有价值的底物。
