Bacillus subtilis deoxyuridinetriphosphatase and its bacteriophage PBS2-induced inhibitor.
Extracts of Bacillus subtilis contain a deoxyuridinetriphosphatase (dUTPase) activity with a molecular weight of approximately 48,000. The enzyme is maximally active at pH 8.5, being stimulated by Mg2+ and inhibited by EDTA. The enzyme is specific for dUTP among all the natural nucleotides tested, with an apparent Km for dUTP of 2 muM. Bacteriophage PBS2, whose DNA contains uracil instead of thymine, induces upon infection of B. subtilis a new 83,000-dalton protein which inhibits the host's dUTPase. The inhibitor acts immediately and reversibly in vitro to inhibit dUMP production from dUTP. The inhibitor's action is maximal in dUTPase assays performed at pH 6 to 7, and is minimal at pH 9.7. The inhibitor seems to form a higher molecular weight complex with the B. subtilis dUTPase. Increasing the pH of the medium for PBS2 infection from pH 7 to pH 8.85 caused a dramatic decrease in the synthesis of phage DNA and progeny phage. The newly synthesized DNA had an altered thymine/uracil ratio, being increased from less than 0.03 to greater than 1.0. We propose that infection at high pH prevents the PBS2-induced dUTPase inhibitor from blocking the B. subtilis dUTPase activity, thereby allowing the degradation of dUTP and the synthesis of dTTP (both of which are DNA polymerase substrates), so that thymine replaces some of the uracil normally found in PBS2 DNA.
D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology*; D001426:Bacterial Proteins / Q000502:physiology*; D001435:Bacteriophages / Q000187:drug effects / Q000502:physiology*; D002701:Chloramphenicol / Q000494:pharmacology; D004279:DNA, Viral / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D003854:Deoxyribonucleotides; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011755:Pyrophosphatases / Q000378:metabolism*; D012293:Rifampin / Q000494:pharmacology; D014500:Uracil Nucleotides; D014764:Viral Proteins / Q000502:physiology*
D001426:Bacterial Proteins; D004279:DNA, Viral; D003854:Deoxyribonucleotides; D014500:Uracil Nucleotides; D014764:Viral Proteins; D003609:Dactinomycin; D002701:Chloramphenicol; D004492:Edetic Acid; D011755:Pyrophosphatases; D008274:Magnesium; D012293:Rifampin