Restriction modification (RM) systems are composed of a restriction endonuclease and a DNA methyltransferase, and provide bacteria with protection against non-self DNA (1
). In most cases, the self DNA is marked by methylation, introduced in a sequence-specific manner by the methyltransferase. Non-self DNA, which lacks the methylation mark, is cleaved by the endonuclease. However, the rule that self, but not non-self DNA is modified, is not universal. Presumably, under the pressure from RM systems, some phages have evolved to incorporate a wide variety of modified DNA bases into their genomes. In a further escalation of the ‘arms race’ between bacteria and their pathogens, this has then led to the evolution of modification-dependent restriction enzymes, which treat unmodified DNA as ‘self’ and modified DNA as ‘non-self’.
Modification-dependent restriction enzymes are a very diverse group, and in the current classification of nucleases fall either into the type IIM (where ‘M’ stands for modification-dependent) or type IV class, depending on subunit composition, biochemical properties and specificity of cleavage (2
). Type IV McrA belongs to the ββα-Me (or HNH) superfamily of nucleases, and is, at least in vivo
, specific for the Ym5CGR target sequence (where Y is C or T and R is G or A) (3
). Type IV McrBC is an oligomeric protein complex, which recognizes pairs of Rm5C sites separated by 40–2000
bp of non-specific DNA. The enzyme consists of the GTPase McrB, which is also required for the recognition of methylation, and the McrC subunit that harbors a PD-(D/E)XK motif and catalyzes DNA cleavage (4
). Type IV Mrr is a cryptic endonuclease thought to act on modified adenines, whose activity appears to be triggered by some forms of stress (6
). The enzyme causes genotoxicity when expressed in bacteria containing certain type III DNA m6A methyltransferases. A strong anti-correlation between close homologues of Mrr and these methyltransferases has been observed in proteobacterial genomes (7
). Some enzymes related to Mrr, including the recently characterized R.MspJI, have been shown to cleave DNA that is methylated on cytosines (8
). There are also many other methylation-specific activities (e.g. R.AoxI, R.BisI, R.BlsI, R.GlaI, R.GluI, R.KroI and R.PscI), which have not yet been ascribed to molecularly defined complexes and their domain composition and mechanism of cleavage remains unknown.
R.DpnI is the best known modification-dependent restriction endonuclease that recognizes methylated adenines rather than cytosines. The enzyme was originally isolated from Streptococcus
). In vivo
, it protects bacteria against phages that have been propagated on Dam+
hosts. This is possible thanks to its specificity for Gm6ATC, the product of Dam methylation. In vitro
, R.DpnI cleaves GATC sites efficiently when these are fully methylated (i.e. in both strands) and also hemi-methylated, when the enzyme is used in high concentration. It is a blunt end cutter that cleaves DNA in the middle of the recognition sequence (10–12
), rather than distantly from it like many other modification-dependent restriction endonucleases. Due to this methylation dependence, R.DpnI has been classified as a type IIM enzyme. Unlike most other type II restriction endonucleases, it is a monomer in solution (10
). Due to its unusual properties, it is widely used in biotechnological applications. A standard site-directed mutagenesis strategy (Stratagene) exploits the enzyme to remove a non-mutated template that has been propagated in a Dam+
host, but not a PCR product carrying the mutation. The preference of R.DpnI for fully methylated over hemi-methylated sites has been used to create ‘ultra-rare’ cutting enzymes by combining it with suitably chosen m6A methyltransferases (13–15
). R.DpnI is also useful for studies of bacterial DNA replication, because digestion with low amounts of the enzyme cleaves all but freshly replicated DNA (12
Despite the widespread use of R.DpnI in biotechnology and the interest in other modification-dependent enzymes, which could be applied to studies of epigenetic modifications of eukaryotic DNA, the present understanding of this group of enzymes is very limited. In particular, crystal or NMR structures have not been reported, and the mechanisms of modification-dependent cleavage remain mysterious. In a first step towards a better understanding of methylation dependent cleavage, we present a detailed biochemical characterization of R.DpnI and its co-crystal structure with fully methylated substrate DNA.