Host specificity of DNA produced by Escherichia coli: XV. The role of nucleotide methylation in in vitro B-specific modification

Abstract

It is shown that in vitro Escherichia coli strain B-specific modification of the replicative form of bacteriophage fd DNA is accompanied by the methylation of certain adenine moieties to form N-6-methyladenine. The reaction follows first order kinetics and saturation is reached when about four adenines are methylated per replicative form. No methyl groups are transferred to B-modified DNA. The replicative form of a one step mutant of fd, which has a reduced sensitivity towards B-specific restriction, has lost two of the four methyl acceptor sites. The replicative form of a second step mutant, which is not subject to B-specific restriction, is completely refractory to methylation by the modification enzyme. It is therefore concluded that the B-modification and the B-restriction enzyme react with the same sites on the substrate DNA and that the replicative form of wild type fd has two such sites. The number of N-6-methyladenines per B-specificity site of fully modified double-stranded DNA is two.