Published online 7 December 2011 Nucleic Acids Research, 2012, Vol. 40, No. 4 e29 doi:10.1093/nar/gkr1146 Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing Tyson A. Clark1, Iain A. Murray2, Richard D. Morgan 2 , Andrey O. Kislyuk 1 , Kristi E. Spittle1, Matthew Boitano 1 , Alexey Fomenkov2, Richard J. Roberts2,* and Jonas Korlach1,* 1Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025 and 2New England Biolabs, 240 County Road, Ipswich, MA 01938, USA Received August 15, 2011; Revised October 30, 2011; Accepted November 10, 2011 ABSTRACT DNA methylation is the most common form of DNA modification in prokaryotic and eukaryotic genomes. We have applied the method of single- molecule, real-time (SMRT ) DNA sequencing that is capable of direct detection of modified bases at single-nucleotide resolution to characterize the spe- cificity of several bacterial DNA methyltransferases (MTases). In addition to previously described SMRT sequencing of N6-methyladenine and 5-methylcytosine, we show that N4-methylcytosine also has a specific kinetic signature and is therefore identifiable using this approach. We demonstrate for all three prokaryotic methylation types that SMRT sequencing confirms the identity and position of the methylated base in cases where the MTase specificity was previously established by other methods. We then applied the method to determine the sequence context and methylated base identity for three MTases with unknown specificities. In addition, we also find evidence of unanticipated MTase promiscuity with some enzymes apparently also modifying sequences that are related, but not identical, to the cognate site. INTRODUCTION Methylation of DNA bases, catalyzed by DNA methyltransferases (MTases), is the most abundant form of post-replicative DNA modiﬁcation found in the genomes of prokaryotic and higher eukaryotic organisms. Three functional classes of MTases have been identiﬁed in bacteria and archaea. Two of these transfer a methyl group from S-adenosyl-L-methionine (SAM) to the exocyclic amino groups of adenine and cytosine bases in duplex DNA, yielding N6-methyladenine (m6A) and N4-methylcytosine (m4C), respectively (1). A third, and mechanistically distinct, class transfers the methyl group of SAM to C5 of cytosine to produce 5-methylcytosine (m5C) (2,3). Most bacterial and archaeal MTases are associated with sequence-speciﬁc restriction-modiﬁcation (RM) systems that protect the prokaryotic cell from invasion by DNA bacteriophages, and examples have been identiﬁed that recognize several hundred distinct DNA sequences (4). However, some well-characterized prokaryotic MTases do not appear to be associated with a cognate restriction endonuclease (REase) and some of these ‘orphan’ MTases perform different cellular functions. For example, the product of the Escherichia coli deoxyadenosine methyl- transferase (dam) gene, M.EcoKDam, which modiﬁes adenine residues in the sequence 50-GATC-30, is involved in both chromosomal replication initiation and in the maintenance of genomic integrity [reviewed earlier (5)]. Most eukaryotic MTases are of the m5C class and are related to their prokaryotic equivalents through a common reaction mechanism that is reﬂected in the con- servation of tertiary structural elements within the enzyme active sites (6). Mammalian m5C-MTase activity is pre- dominantly targeted to CpG dinucleotides and three dif- ferent enzymes (DNMT1, DNMT3A and 3B) are known (7). Such DNA methylation is a key component of the epigenetic control of gene expression [reviewed earlier (8)]. CpG methylation is also a key player in genomic im- printing and in female X-inactivation (9,10). An addition- al modiﬁed base, 5-hydroxymethylcytosine (5-hmC), was presumed to be a product of DNA damage (11), but has *To whom correspondence should be addressed. Tel: +(978) 380 7405; Fax: +(978) 380 7406; Email: roberts@neb.com Correspondence may also be addressed to Jonas Korlach. Tel: +(650) 521 8006; Fax: +(650) 323 9420; Email: jkorlach@paciﬁcbiosciences.com The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.