ANALYSIS OF INHIBITORS OF BACTERIOPHAGE-T4 DNA-POLYMERASE

Bacteriophage T4 DNA polymerase was inhibited by butylphenyl nucleotid es, aphidicolin and pyrophosphate analogs, but with lower sensitivitie s than other members of the B family DNA polymerases. The nucleotides N-2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butylanilino)dATP (BuAd ATP) inhibited T4 DNA polymerase with competitive K-i values of 0.82 a nd 0.54 mu M with respect to dGTP and dATP, respectively. The same com pounds were more potent inhibitors in truncated assays lacking the com petitor dNTP, displaying apparent K-i values of 0.001 and 0.0016 mu M, respectively. BuPdGTP was a substrate for T4 DNA polymerase, and the resulting 3'-BuPdG-primer:template was bound strongly by the enzyme. E ach of the non-substrate derivatives, BuPdGDP and BuPdGMPCH(2)PP, inhi bited T4 DNA polymerase with similar potencies in both the truncated a nd variable competitor assays. These results indicate that BuPdGTP inh ibits T4 DNA polymerase by distinct mechanisms depending upon the assa y conditions. Reversible competitive inhibition predominates in the pr esence of dGTP, and incorporation in the absence of dGTP leads to pote nt inhibition by the modified primer:template. The implications of the se findings for the use of these inhibitors in the study of B family D NA polymerases is discussed.