Inhibition of a DNA Polymerase from Bacillus subtilis by Hydroxyphenylazopyrimidines

Abstract

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...

RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

“…HPUra has been a very important tool for investigating the involvement of DNA replication in a number of different DNA transactions in vivo (see, e.g., references 10, 48, 49, and 54). Prior work suggested that the hydrazine congener (H 2 -HPUra) is the active form that inhibits DNA synthesis (37,47,50,51). We were unable to synthesize H 2 -HPUra and could obtain only the oxidized form (HPUra).…”

Deoxyribonucleic Acid Polymerases from Yeast

“…Cytosine-arabinoside is known to affect DNA replication and to inhibit the DNA polymerases I1 and Ill but not I of E. coli [27] and Bacillus subtilis [28] as well as the high-molecular-weight DNA polymerases of animal cells [29,30]. I have studied the effect of cytosine-arabinoside triphosphate on the activity of the two yeast DNA polymerases.…”

Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme

“…Indeed, addition of dGTP to the enzymatic Pol IIIC assay increased the IC 50 s of the AU compounds (data not shown), which is consistent with the competitive mode of action of this compound class (25). (iii) It has been reported that the S. aureus Pol IIIC activity is lowered from ϳ480 to ϳ120 nucleotides/s in in vitro assays, in which the accessory proteins present in the bacterial cell, e.g., the ␤ sliding clamp, are lacking (11). Thus, it might be that the molecules are able to inhibit the slower activity of the Pol IIIC enzyme in the in vitro assay but are not potent enough to inhibit the enzymatic activity to the same extent inside a bacterial cell in the presence of dGTP.…”