Inhibition of a Discrete Bacterial DNA Polymerase by 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenylazo)-uracil and 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenylazo-)-isocytosine

6-(p-HYDROXYPHENYLAZO)-URACIL (HPUra) specifically inhibits the semi-conservative replication of DNA in Gram-positive bacteria^1–3. We have reported that HPUra inhibits ATP-dependent polymerization of deoxyribonucleotides in vitro in toluene-treated B. subtilis⁴. Further studies of the effect of HPUra and its amino analogue, HPIsocytosine (6-(p-hydroxyphenylazo)-2-amino, 4-keto pyrimidine), in toluene-treated B. subtilis have provided considerable information on the mechanism of arylazopyrimidine action. First, HPUra and HPIsocytosine do not inhibit DNA synthesis unless they first are reduced to their colourless, hydrazo forms (refs. 4 and 5 and Mackenzie, Wright and Brown, unpublished results). Second, the inhibitory action of reduced HPUra and that of reduced HPIsocytosine are completely antagonized, respectively, by dGTP and dATP⁵. Third, drug-resistant mutants have been isolated which catalyse drug-resistant DNA synthesis following their permeabilization with toluene. These observations suggest that reduced HPUra and HPIsocytosine inhibit DNA replication by interfering competitively with the enzymatic polymerization of specific purine deoxyribonucleotides. We examined, therefore, cell free preparations of ^{B. subtilis} in an effort to identify a discrete DNA polymerase as the site of drug action. We report here experiments with crude and partially fractionated extracts of DNA polymerase I-deficient mutants which indicate the existence of at least one drug sensitive polymerase. Bazill and Gross⁶ have independently isolated chromatographically discrete HPUra-sensitive polymerases from extracts of B. subtilis.