The antimicrobial agent, 6-(p-hydroxyphenylazo)-uracil, specifically inhibits DNA polymerase III of Bacillus subtilis with a Ki of less than 1 pM. The inhibition requires prior reduction of the drug, is reversible, and is competitive with dGTP. High amounts of dATP prevent inhibition by the closely related drug, 6-(p-hydroxyphenylazo)-isocytosine. A model is presented in which the inhibitors base-pair with the template while part of a ternary complex with the enzyme. These results imply that DNA polymerase III of B. subtilis is necessary for chromosomal replication.The antimicrobial agent, 6-(p-hydroxyphenylazo)-uracil (HPUra), specifically inhibits DNA synthesis in several Gram-positive bacteria (1). In Bacillus subtilis, the inhibition is limited to replicative synthesis, while repair synthesis and DNA synthesis directed by several bacteriophages is unimpaired (2, 3). Semiconservative DNA synthesis in toluenetreated B. subtilis cells is inhibited by HPUra in the presence of reducing agents (4).Recently, we described a mutant of B. subtilis lacking the DNA polymerase isolated by Okazaki and Kornberg (polymerase I) (5, 6). In these polA -cells, the residual DNA-polymerizing activity has markedly different properties from polymerase I (5). During the analysis of this residual activity we have isolated two additional DNA polymerases that we call polymerases II and III, in order of discovery. The three B. subtilis polymerases are similar to the correspondingly numbered Escherichia coli enzymes (7-10), but there are clear differences (11) Drugs. HPUra and 6-(p-hydroxyphenylazo)-2-amino-4-hydroxypyrimidine (HPIso) were generously provided by Dr. Bernard Langley of Imperial Chemical Industries, Ltd., Macclesfield, England. The removal of minor contaminants from HPUra by paper or column chromatography (1) did not alter the inhibitory activity of the drug. To convert the drug to an active inhibitor, 2.0 mM HPUra was treated with 25 mM dithiothreitol in 50 mM Tris* HCl (pH 7.5) at 370 for 21 min; longer incubation times result in a gradual loss of inhibitory activity. During the activation reaction the red color of the drug is lost at a rate that is directly proportional to the concentration of both HPUra and dithiothreitol; the second-order rate constants are 4.4 and 6.5 M-l min' at 300 and 370, respectively. Although the activated form (absorption maximum at 260-265 nm) can be readily purified from dithiothreitol and inactive drug by DEAE-cellulose chromatography, it rapidly reoxidizes in air to the colored form. Thus, for the experiments in this report, the activated drug was used without purification, and its concentration was assumed to be equal to the input inactive form.Enzymes. Deoxyribonuclease I was purchased from Worthington Biochemical Corp. E. coli DNA polymerase I, Fraction 7 (12), and T4 DNA polymerase, Fraction VII (13), were gifts of Dr. P. T. Englund. B. subtilia polymerase I was purified through the phosphocellulose chromatography step by the published method (6). Purification and properties of...

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Of six deoxyribonucleic acid repair mutants of Bacillus subtilis assayed for deoxyribonucleic acid polymerase, only the methyl methanesulfonate-sensitive and ultraviolet light-sensitive mutant JBI-49(59) has impaired polymerase activity. Extracts prepared by sonic treatment or gentle lysis had about 10% of the wild-type activity with poly d(A-T), an alternating copolymer of deoxyadenylate and deoxythymidylate, used as template. The sensitivity to methyl methanesulfonate and ultraviolet light and the low level of polymerase activity transformed and reverted together, indicating that the two characteristics are a pleiotropic manifestation of a single mutation. Mixed extract and kinetic experiments mitigated against an altered nuclease activity as the enzymatic consequence of the mutation. Also, the mutant and wild type activities were stimulated equally by Escherichia coli exonuclease III. The residual activity in the mutant showed several differences from the wild-type activity: it purified differently, was more sensitive to sulfhydryl reagents, and displayed a different template specificity. We tentatively conclude that either the mutation in JBI-49(59) has introduced a qualitative as well as a quantitative change in the polymerase or the wild type contains two distinct polymerases, one of which is missing in the mutant.

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