title: 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 (m)CNNR (R = G/A) sites and cleaves DNA at fixed distances (N(12)/N(16)) away from the modified cytosine at the 3' side (or N(9)/N(13) 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.
authors.affiliation: New England BioLabs, Inc., 240 County Road, Ipswich, MA, 01938, USA. zhengy@neb.com
authors.forename: Yu
authors.initials: Y
authors.lastname: Zheng
authors.forename: Devora
authors.initials: D
authors.lastname: Cohen-Karni
authors.forename: Derrick
authors.initials: D
authors.lastname: Xu
authors.forename: Hang Gyeong
authors.initials: HG
authors.lastname: Chin
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Wilson
authors.forename: Sriharsa
authors.initials: S
authors.lastname: Pradhan
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000145:classification; D016678:Genome; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D004247:DNA; D004262:DNA Restriction Enzymes; C070141:Mrr protein, E coli
references.citation: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W649-52
references.pmid: 17452345
references.citation: Biochim Biophys Acta. 1975 Jun 16;395(2):109-19
references.pmid: 1138935
references.citation: BMC Mol Biol. 2008 Jan 15;9:7
references.pmid: 18194583
references.citation: Gene. 2001 Apr 18;267(2):183-91
references.pmid: 11313145
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D237-40
references.pmid: 17135202
references.citation: Protein Expr Purif. 2008 Nov;62(1):98-103
references.pmid: 18662788
references.citation: Annu Rev Microbiol. 1980;34:137-58
references.pmid: 7002022
references.citation: Nucleic Acids Res. 2007;35(6):1992-2002
references.pmid: 17341463
references.citation: J Bacteriol. 1991 Aug;173(16):5207-19
references.pmid: 1650347
references.citation: J Bacteriol. 1987 Jul;169(7):3243-50
references.pmid: 3036779
references.citation: Proc Natl Acad Sci U S A. 1986 Dec;83(23):9070-4
references.pmid: 3024165
references.citation: Mol Microbiol. 1992 May;6(9):1079-86
references.pmid: 1316984
references.citation: Mol Microbiol. 2005 Dec;58(5):1381-91
references.pmid: 16313623
references.citation: J Mol Biol. 1992 May 20;225(2):327-48
references.pmid: 1317461
references.citation: J Biol Chem. 1975 Jun 10;250(11):4060-66
references.pmid: 236309
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D234-6
references.pmid: 19846593
references.citation: Nature. 2009 Nov 19;462(7271):315-22
references.pmid: 19829295
references.citation: J Biol Chem. 2002 Feb 8;277(6):4024-33
references.pmid: 11729187
references.citation: Science. 2009 May 15;324(5929):930-5
references.pmid: 19372391
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D269-70
references.pmid: 17202163
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10564-9
references.pmid: 9724743