The non-specific adenine DNA methyltransferase M.EcoGII.

• Type of Material: Article
• Authors: Iain A Murray, New England Biolabs; Richard D Morgan, New England Biolabs; Yvette Luyten, New England Biolabs; Alexey Fomenkov, New England Biolabs; Ivan R. Corrêa, New England Biolabs; Nan Dai, New England Biolabs; Mohammed B Allaw, Emory University; Xing Zhang, Emory University; Xiaodong Cheng, Emory University; Richard J Roberts, New England Biolabs
• Language: English
• Date: 2018-01-25
• Publisher: Oxford University Press
• Publication Version: Final Published Version
• Copyright Statement: © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
• License: Creative Commons Attribution-NonCommercial 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1093/nar/gkx1191
• Title of Journal or Parent Work: Nucleic Acids Research
• ISSN: 0305-1048
• Volume: 46
• Issue: 2
• Start Page: 840
• End Page: 848
• Grant/Funding Information: New England Biolabs; National Institutes of Health [R44 GM100560 to R.J.R., R01 GM049245 to X.C.]. Funding for open access charge: New England BioLabs Inc., Ipswich, MA, USA.
• Research Categories: Biology, Genetics; Biology, Molecular

Abstract

We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo - using a high copy pRRS plasmid vector and a methylation-deficient E. coli host-extensive in vivo adenine methylation activity is revealed. M.EcoGII methylates adenine residues in any DNA sequence context and this activity extends to dA and rA bases in either strand of a DNA:RNA-hybrid oligonucleotide duplex and to rA bases in RNAs prepared by in vitro transcription. Using oligonucleotide and bacteriophage M13mp18 virion DNA substrates, we find that M.EcoGII also methylates single-stranded DNA in vitro and that this activity is only slightly less robust than that observed using equivalent double-stranded DNAs. In vitro assays, using purified recombinant M.EcoGII enzyme, demonstrate that up to 99% of dA bases in duplex DNA substrates can be methylated thereby rendering them insensitive to cleavage by multiple restriction endonucleases. These properties suggest that the enzyme could also be used for high resolution mapping of protein binding sites in DNA and RNA substrates.