Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
Doctoral School in Biology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary.
PLoS One. 2018 May 10;13(5):e0197232. doi: 10.1371/journal.pone.0197232. eCollection 2018.
The highly similar prokaryotic DNA (cytosine-5) methyltransferases (C5-MTases) M.MpeI and M.SssI share the specificity of eukaryotic C5-MTases (5'-CG), and can be useful research tools in the study of eukaryotic DNA methylation and epigenetic regulation. In an effort to improve the stability and solubility of complementing fragments of the two MTases, genes encoding circularly permuted (CP) variants of M.MpeI and M.SssI were created, and cloned in a plasmid vector downstream of an arabinose-inducible promoter. MTase activity of the CP variants was tested by digestion of the plasmids with methylation-sensitive restriction enzymes. Eleven of the fourteen M.MpeI permutants and six of the seven M.SssI permutants had detectable MTase activity as indicated by the full or partial protection of the plasmid carrying the cpMTase gene. Permutants cp62M.MpeI and cp58M.SssI, in which the new N-termini are located between conserved motifs II and III, had by far the highest activity. The activity of cp62M.MpeI was comparable to the activity of wild-type M.MpeI. Based on the location of the split sites, the permutants possessing MTase activity can be classified in ten types. Although most permutation sites were designed to fall outside of conserved motifs, and the MTase activity of the permutants measured in cell extracts was in most cases substantially lower than that of the wild-type enzyme, the high proportion of circular permutation topologies compatible with MTase activity is remarkable, and is a new evidence for the structural plasticity of C5-MTases. A computer search of the REBASE database identified putative C5-MTases with CP arrangement. Interestingly, all natural circularly permuted C5-MTases appear to represent only one of the ten types of permutation topology created in this work.
高度相似的原核 DNA（胞嘧啶-5）甲基转移酶（C5-MTases）M.MpeI 和 M.SssI 具有真核 C5-MTases（5'-CG）的特异性，并且可以成为研究真核 DNA 甲基化和表观遗传调控的有用研究工具。为了提高两种 MTases 互补片段的稳定性和可溶性，构建了编码环状排列（CP）变体的 M.MpeI 和 M.SssI 的基因，并将其克隆到阿拉伯糖诱导启动子的质粒载体下游。通过用甲基化敏感的限制酶消化质粒来测试 CP 变体的 MTase 活性。在 14 个 M.MpeI 变构体中有 11 个和 7 个 M.SssI 变构体中的 6 个具有可检测的 MTase 活性，这表明携带 cpMTase 基因的质粒完全或部分受到保护。在新的 N-末端位于保守模体 II 和 III 之间的变构体 cp62M.MpeI 和 cp58M.SssI 中，活性最高。cp62M.MpeI 的活性可与野生型 M.MpeI 的活性相媲美。根据分割位点的位置，可以将具有 MTase 活性的变构体分类为十种类型。尽管大多数变构位点被设计为位于保守模体之外，并且在细胞提取物中测量的变构体的 MTase 活性在大多数情况下远低于野生型酶，但与 MTase 活性兼容的环状排列拓扑结构的高比例是值得注意的，这是 C5-MTases 结构可塑性的新证据。对 REBASE 数据库的计算机搜索确定了具有 CP 排列的推定 C5-MTases。有趣的是，所有天然的环状排列的 C5-MTases 似乎都只代表了本工作中创建的十种变构拓扑类型之一。
