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J Mol Biol. 2007 Feb 23;366(3):768-78. Epub 2006 Nov 21.

A type IV modification dependent restriction nuclease that targets glucosylated hydroxymethyl cytosine modified DNAs.

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1
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201-1503, USA.

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.

PMID:
17188297
PMCID:
PMC1855630
DOI:
10.1016/j.jmb.2006.11.051
[Indexed for MEDLINE]
Free PMC Article
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