MspJI is a novel modification-dependent restriction endonuclease that cleaves at a fixed distance away from the modification site. Here, we present the biochemical characterization of several MspJI homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI. All of the enzymes specifically recognize cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleave at a constant distance (N 12 ∕N 16 ) away from the modified cytosine. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. By cleaving on both sides of fully modified CpG sites, they allow the extraction of 32-base long fragments around the modified sites from the genomic DNA. These enzymes provide powerful tools for direct interrogation of the epigenome. For example, we show that RlaI, an enzyme that prefers m CWG but not m CpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. In addition, we demonstrate that deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome. Altogether, the MspJI family of enzymes represent appealing tools of choice for method development in DNA epigenetic studies. 5-methylcytosine | methylomeM odified DNA bases appear in genomic DNAs in all domains of life, spanning the evolutionary distance from viruses to eukaryotic species. DNA base modifications vary in form and genomic location enriching the information content encoded by genomes. The biological role of base modifications varies, ranging from protection against restriction endonucleases in bacteria and bacteriophages to transcriptional regulation in mammals. In prokaryotes, DNA methyltransferases in restriction-modification systems modify the host genomic DNA, so that restriction endonucleases can target foreign DNA and protect the host cell from invaders (1). However, a few bacteriophages respond by incorporating modified bases into their genomes as a way to block restriction endonuclease cleavage (2). For example, in Xanthomonas oryzae phage XP12, all cytosines exist in the form of 5-methylcytosine (5mC) (3). Another example is the well-studied T4 phage, in which 5-hydroxymethylcytosine (5hmC) is incorporated into the DNA during replication and additional glucosyltransferases further modify all 5hmC to glucosylated-hydroxymethylcytosine (5ghmC). T4 genomic DNA containing 5ghmC is resistant to cleavage by most restriction endonucleases, with the exception of Type IV modification-dependent endonucleases (4, 5).Several different types of modification-dependent endonucleases are found in prokaryotes. For example, N6-adenosine methylation is recognized by a few known enzymes, e.g., DpnI (G m ATC). A group of sequence-specific cytosine methylationdependent restriction endonucleases including GlaI (G m CG m C), BisI (G m CNGC), etc., have been reported recently, which cleave within the recognition site in a Type IIP-like ...

Mrr superfamily of homologous genes in microbial genomes restricts modified DNA in vivo. However, their biochemical properties in vitro have remained obscure. Here, we report the experimental characterization of MspJI, a remote homolog of Escherichia coli’s Mrr and show it is a DNA modification-dependent restriction endonuclease. Our results suggest MspJI recognizes mCNNR (R = G/A) sites and cleaves DNA at fixed distances (N12/N16) away from the modified cytosine at the 3′ side (or N9/N13 from R). Besides 5-methylcytosine, MspJI also recognizes 5-hydroxymethylcytosine but is blocked by 5-glucosylhydroxymethylcytosine. Several other close homologs of MspJI show similar modification-dependent endonuclease activity and display substrate preferences different from MspJI. A unique feature of these modification-dependent enzymes is that they are able to extract small DNA fragments containing modified sites on genomic DNA, for example ∼32 bp around symmetrically methylated CG sites and ∼31 bp around methylated CNG sites. The digested fragments can be directly selected for high-throughput sequencing to map the location of the modification on the genomic DNA. The MspJI enzyme family, with their different recognition specificities and cleavage properties, provides a basis on which many future methods can build to decode the epigenomes of different organisms.

A type IIG restriction endonuclease, RM.BpuSI from Bacillus pumilus, has been characterized and its X-ray crystal structure determined at 2.35Å resolution. The enzyme is comprised of an array of 5-folded domains that couple the enzyme's N-terminal endonuclease domain to its C-terminal target recognition and methylation activities. The REase domain contains a PD-x15-ExK motif, is closely superimposable against the FokI endonuclease domain, and coordinates a single metal ion. A helical bundle domain connects the endonuclease and methyltransferase (MTase) domains. The MTase domain is similar to the N6-adenine MTase M.TaqI, while the target recognition domain (TRD or specificity domain) resembles a truncated S subunit of Type I R–M system. A final structural domain, that may form additional DNA contacts, interrupts the TRD. DNA binding and cleavage must involve large movements of the endonuclease and TRD domains, that are probably tightly coordinated and coupled to target site methylation status.