Modification and restriction of T-even bacteriophages. In vitro degradation of deoxyribonucleic acid containing 5-hydroxymethylctosine.
Using the single-stranded circular DNA of bacteriophage fd as template, double-stranded circular DNA has been prepared in vitro with either 5-hydroxymethylcytosine ([hmdC]DNA) or cytosine ([dC]DNA) in the product strand. Extracts prepared from Escherichia coli cells restrictive to T-even phage containing nonglucosylated DNA degrade [hmdC]DNA to acid-soluble material in vitro, but do not degrade [dC]dna. In contrast, extracts prepared from E. coli K12 rglA- rglB-, a strain permissive to T-even phage containing nonglucosylated DNA, do not degrade [hmdC]DNA or [dC]DNA. In addition, glucosylation of the [hmdC]DNA renders it resistant to degradation by extracts from restrictive strains. The conversion of [hmdC]DNA to acid-soluble material in vitro consists of an HmCyt-specific endonucleolytic cleavage requiring the presence of the RglB gene product to form a linear molecule, followed by a non-HmCyt-specific hydrolysis of the linear DNA to acid-soluble fragments, catalyzed in part by exonuclease V. The RglB protein present in extracts of E. coli K12 rglA- rglB+ has been purified 200-fold by complementation with extracts from E. coli K12 rglA- rglB-. The purified RglB protein does not contain detectable HmCyt-specific endonuclease or exonuclease activity. In vitro endonucleolytic cleavage of [hmdC]DNA thus requires additional factors present in cell extracts.
D044503:5-Methylcytosine / Q000031:analogs & derivatives; D003090:Coliphages / Q000378:metabolism*; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D007700:Kinetics; D009154:Mutation; D010641:Phenotype; D013045:Species Specificity; D013698:Templates, Genetic
D004270:DNA, Circular; D004279:DNA, Viral; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004262:DNA Restriction Enzymes