Inhibition of bacterial DNA replication by 6-( p-Hydroxyphenylazo)-uracil: Differential effect on repair and semi-conservative synthesis in Bacillus subtilis

Abstract

6-( p-Hydroxyphenylazo)-uracil is a reversible, selective inhibitor of semi-conservative replication of DNA in Bacillus subtilis and other Gram-positive bacteria. The effects of HPUra † † Abbreviations used: HPUra, 6-( p-hydroxyphenylazo)-uracil; BrdUrd, 5-bromo-2′-deoxyinidine. were examined on the replication of DNA following ultraviolet irradiation of B. subtilis 168 thy − ind −. Ultraviolet irradiation stimulated a form of DNA synthesis which was resistant to HPUra. Drug-resistant synthesis increased in rate with increasing doses of ultraviolet irradiation and was inhibited by acriflavin and actinomycin D, inhibitors of excision-repair of bacterial DNA. Evidence is presented which suggests that the HPUra-resistant synthesis was not a phage-specific process resulting from ultraviolet induction of defective prophage. The product of ultraviolet-stimulated DNA synthesis was labelled with radioactive bromodeoxyuridine and analysed by equilibrium centrifugation in cesium chloride. The product was typical of repair-replicated DNA; it was detected only in DNA of irradiated cells and was synthesized in approximately the same amounts in the presence and absence of HPUra. The bromodeoxyuridine-labelled product of ultraviolet-stimulated synthesis banded close to the density of native B. subtilis DNA; sonic shear of this product increased its buoyant density slightly, suggesting that the bulk of bromodeoxyuridine was in short chains. Centrifugation of the DNA after alkaline denaturation indicated that these chains were linked covalently to parental DNA. These results suggested (1), that B. subtilis 168 thy − ind − was capable of repair replication of ultraviolet-damaged DNA and (2), that this process, unlike semi-conservative replication, was refractory to HPUra treatment.