title: Characterization of the 5-hydroxymethylcytosine-specific DNA restriction endonucleases.
abstract: In T4 bacteriophage, 5-hydroxymethylcytosine (5hmC) is incorporated into DNA during replication. In response, bacteria may have developed modification-dependent type IV restriction enzymes to defend the cell from T4-like infection. PvuRts1I was the first identified restriction enzyme to exhibit specificity toward hmC over 5-methylcytosine (5mC) and cytosine. By using PvuRts1I as the original member, we identified and characterized a number of homologous proteins. Most enzymes exhibited similar cutting properties to PvuRts1I, creating a double-stranded cleavage on the 3' side of the modified cytosine. In addition, for efficient cutting, the enzymes require two cytosines 21-22-nt apart and on opposite strands where one cytosine must be modified. Interestingly, the specificity determination unveiled a new layer of complexity where the enzymes not only have specificity for 5-β-glucosylated hmC (5βghmC) but also 5-α-glucosylated hmC (5αghmC). In some cases, the enzymes are inhibited by 5βghmC, whereas in others they are inhibited by 5αghmC. These observations indicate that the position of the sugar ring relative to the base is a determining factor in the substrate specificity of the PvuRts1I homologues. Lastly, we envision that the unique properties of select PvuRts1I homologues will permit their use as an additive or alternative tool to map the hydroxymethylome.
authors.affiliation: New England Biolabs Inc., 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Janine G
authors.initials: JG
authors.lastname: Borgaro
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Zhu
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D017122:Bacteriophage T4 / Q000235:genetics; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D053837:DNA Cleavage; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004279:DNA, Viral; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D005964:Glucosyltransferases; D004262:DNA Restriction Enzymes; C024369:endodeoxyribonuclease Rts1