Analysis of inhibitors of bacteriophage T4 DNA polymerase.
Bacteriophage T4 DNA polymerase was inhibited by butylphenyl nucleotides, aphidicolin and pyrophosphate analogs, but with lower sensitivities than other members of the B family DNA polymerases. The nucleotides N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butylanilino)dATP (BuAdATP) inhibited T4 DNA polymerase with competitive Ki values of 0.82 and 0.54 microM with respect to dGTP and dATP, respectively. The same compounds were more potent inhibitors in truncated assays lacking the competitor dNTP, displaying apparent Ki values of 0.001 and 0.0016 microM, respectively. BuPdGTP was a substrate for T4 DNA polymerase, and the resulting 3'-BuPdG-primer:template was bound strongly by the enzyme. Each of the non-substrate derivatives, BuPdGDP and BuPdGMPCH2PP, inhibited T4 DNA polymerase with similar potencies in both the truncated and variable competitor assays. These results indicate that BuPdGTP inhibits T4 DNA polymerase by distinct mechanisms depending upon the assay conditions. Reversible competitive inhibition predominates in the presence of dGTP, and incorporation in the absence of dGTP leads to potent inhibition by the modified primer:template. The implications of these findings for the use of these inhibitors in the study of B family DNA polymerases is discussed.
Khan
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NN
Department of Pharmacology, University of Massachusetts Medical School, Worcester 01655.
Reha-Krantz
L J
LJ
Wright
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GE
D000227:Adenine Nucleotides / Q000494:pharmacology; D016590:Aphidicolin / Q000031:analogs & derivatives / Q000494:pharmacology; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors
D000227:Adenine Nucleotides; D003848:Deoxyguanine Nucleotides; D006150:Guanine Nucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D016590:Aphidicolin; C040364:N(2)-(4-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate; D004259:DNA-Directed DNA Polymerase