title: A type IV modification dependent restriction nuclease that targets glucosylated hydroxymethyl cytosine modified DNAs.
abstract: The Escherichia coli CT596 prophage exclusion genes gmrS and gmrD were found to encode a novel type IV modification-dependent restriction nuclease that targets and digests glucosylated (glc)-hydroxymethylcytosine (HMC) DNAs. The protein products GmrS (36 kDa) and GmrD (27 kDa) were purified and found to be inactive separately, but together degraded several different glc-HMC modified DNAs (T4, T2 and T6). The GMR enzyme is able to degrade both alpha-glucosy-HMC T4 DNA and beta-glucosyl-HMC T4 DNA, whereas no activity was observed against non-modified DNAs including unmodified T4 cytosine (C) DNA or non-glucosylated T4 HMC DNA. Enzyme activity requires NTP, favors UTP, is stimulated by calcium, and initially produces 4 kb DNA fragments that are further degraded to low molecular mass products. The enzyme is inhibited by the T4 phage internal protein I* (IPI*) to which it was found to bind. Overall activities of the purified GmrSD enzyme are in good agreement with the properties of the cloned gmr genes in vivo and suggest a restriction enzyme specific for sugar modified HMC DNAs. IPI* thus represents a third generation bacteriophage defense against restriction nucleases of the Gmr type.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201-1503, USA.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Bair
authors.forename: Lindsay W
authors.initials: LW
authors.lastname: Black
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D036022:Capsid Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D003090:Coliphages; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000821:virology; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019143:Evolution, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D017900:Myoviridae; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; C035493:DNA, glucosylated; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D009711:Nucleotides; C046947:internal protein I, bacteriophage T4; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; C059680:DNA modification methylase TaqI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes