EaStChem School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh EH9 3JJ, UK.
New England BioLabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Biochem Biophys Res Commun. 2014 Jun 20;449(1):120-5. doi: 10.1016/j.bbrc.2014.04.162. Epub 2014 May 9.
EcoP15I is a Type III DNA restriction and modification enzyme of Escherichia coli. We show that it contains two modification (Mod) subunits for sequence-specific methylation of DNA and one copy of a restriction endonuclease (Res) subunit for cleavage of DNA containing unmethylated target sequences. Previously the Mod2 dimer in the presence of cofactors was shown to use nucleotide flipping to gain access to the adenine base targeted for methylation (Reddy and Rao, J. Mol. Biol. 298 (2000) 597-610.). Surprisingly the Mod2 enzyme also appeared to flip a second adenine in the target sequence, one which was not subject to methylation. We show using fluorescence lifetime measurements of the adenine analogue, 2-aminopurine, that only the methylatable adenine undergoes flipping by the complete Res1Mod2 enzyme and that this occurs even in the absence of cofactors. We suggest that this is due to activation of the Mod2 core by the Res subunit.
EcoP15I 是大肠杆菌的一种 III 型 DNA 限制修饰酶。我们表明，它包含两个用于 DNA 序列特异性甲基化的修饰（Mod）亚基和一个用于切割含有未甲基化靶序列的 DNA 的限制内切酶（Res）亚基。此前，已有研究表明，在辅助因子存在的情况下，Mod2 二聚体通过核苷酸翻转来获得被靶向甲基化的腺嘌呤碱基（Reddy 和 Rao，J. Mol. Biol. 298 (2000) 597-610.）。令人惊讶的是，Mod2 酶似乎也会翻转靶序列中的第二个腺嘌呤，而这个腺嘌呤不受甲基化的影响。我们通过对腺嘌呤类似物 2-氨基嘌呤的荧光寿命测量表明，只有可甲基化的腺嘌呤通过完整的 Res1Mod2 酶发生翻转，即使在没有辅助因子的情况下也是如此。我们认为这是由于 Res 亚基激活了 Mod2 核心。
