Analysis of Host Range Restriction in Escherichia coli Treated with Toluene

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Abstract
Escherichia coli cells treated with toluene replicate DNA when they are provided with deoxyribonucleoside 5'-triphosphates, ATP, Mg(++), and K(+). However, when deoxycytidine 5'-triphosphate is replaced by hydroxymethyl deoxycytidine 5'-triphosphate, incorporation of nucleotides into acid-precipitable material by toluenetreated strains restrictive to nonglucosylated T-even phage is reduced to less than 5% of that normally observed. Even when dCTP is present in the reaction mixture, a similar effect of the hydroxymethyl analogue on DNA replication is observed. In contrast, toluene-treated E. coli K12r6(-)r2,4(-), a strain permissive to the nonglucosylated T-even phage, incorporates hydroxymethyl deoxycytosine into its DNA, and replication proceeds at only a slightly reduced rate in the presence of the hydroxymethyl deoxycytidine 5'-triphosphate. The presence of the hydroxymethyl deoxycytidine 5'-triphosphate in the reaction mixture does not lead to degradation of preexisting DNA of the restrictive host, but it does lead to an irreversible inhibition of further DNA replication; the inhibition is observed only when the hydroxymethyl deoxycytidine 5'-triphosphate is present during replication. Thus phage-specific enzymes are not necessary for the incorporation of hydroxymethylcytosine into phage DNA, and the restrictive mechanism, present in the host cell before infection, can recognize hydroxymethylcytosine residues in its own DNA, as well as the DNA of the T-even phage.