A unique family of Mrr-like modification-dependent restriction endonucleases

Abstract

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.

The MspJI family of modification-dependent restriction endonucleases for epigenetic studies

“…MspJI and its homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI, were synthesized using the overlapping oligonucleotide assembly method as previously described (14). Recombinant enzymes, with or without N-terminal His-tags, were then expressed in the dcm -E. coli strain T7 Express and purified to apparent homogeneity (SI Appendix, Fig.…”

“…We have previously characterized MspJI as a modificationdependent endonuclease that recognizes 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) and cleaves both strands at specific positions (N 12 ∕N 16 ) on the 3′ side of the modified base (14). The activity of the homologs was assessed using a set of modified plasmids or genomic DNA.…”

“…Recently, our group reported the discovery of a unique group of Mrr-like modification-dependent restriction endonucleases, represented by MspJI (14). MspJI recognizes 5mC in the context of m CNNR (R ¼ G or A) and introduces double-stranded breaks at fixed distances (N 12 ∕N 16 from m C) on the 3′ side of the m C, leaving a four-base 5′ overhang.…”

“…Based on our bioinformatic analysis, we have identified a number of MspJI homologs in GenBank (14). In this paper, we present the detailed characterization of additional MspJI homologs.…”

Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria

“…Invading genomes evade restriction by using a multitude of antirestriction strategies to increase their fitness (10,20,35). Bacteria counteract these antirestriction strategies by acquiring additional R-M systems with distinct recognition specificities (36). The continuous selection for the fitness advantage leads to a coevolutionary arms race between the phages and their hosts (10,20).…”