New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
Nucleic Acids Res. 2019 Oct 10;47(18):9761-9776. doi: 10.1093/nar/gkz755.
Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.
依赖修饰的限制性内切酶（MDREs）通常具有单独的催化和依赖修饰的结构域。我们系统地寻找了以前未被表征的融合蛋白，这些融合蛋白具有 PUA 或 DUF3427 结构域和 HNH 或 PD-（D/E）XK 催化结构域。这些酶根据其假定的修饰感应结构域的相似性聚类为几个组。TspA15I（VcaM4I、CmeDI）、ScoA3IV（MsiJI、VcaCI）和 YenY4I 组都具有 PUA 超家族结构域，优先切割含有 5-甲基胞嘧啶或 5-羟甲基胞嘧啶的 DNA。ScoA3V 也具有 PUA 超家族结构域，但属于不同的分支，表现出 6-甲基腺嘌呤刺激的缺口活性。除了少数例外，含有 PUA 超家族结构域的内切酶的 ORF 与 DNA 甲基转移酶的 ORF 不接近，强烈支持内切酶的依赖修饰的活性。尽管存在假定的 PD-（D/E）XK 催化结构域，但含有 DUF3427 结构域的融合蛋白的内切酶活性非常低或没有。然而，它们的表达在大肠杆菌细胞中强烈限制了噬菌体 T4gt。与含有 PUA 结构域的内切酶的 ORF 相反，含有 DUF3427 融合蛋白的 ORF 经常在防御岛上发现，这些基因通常也含有 DNA 甲基转移酶。
