Captan binding to avian myeloblastosis virus reverse transcriptase and its effect on RNase H activity

Summary

The inhibitor captan (N-trichloromethylthio-4-cyclohexen-1,2-dicarboximide) was used to explore the ribonuclease H (RNase H) active site of avian myeloblastosis virus (AMV) reverse transcriptase. Gel permeation chromatography of purified enzyme showed that [^l4C]captan bound to the a subunit in a ratio of 10:1 and to a 32,000 d polypeptide in a ratio of 4:1. Neither the αβ nor the β subunit bound [^l4C]captan. The binding of 5 of the captan molecules was prevented by preincubating enzyme with polynucleotide. Deoxyguanosine triphosphate (dGTP) protected the enzyme against the binding of 4 captan molecules. Each holoenzyme bound 2 molecules of [³H]dGTP in the absence of, and 1 molecule of [³H]dGTP in the presence of 1 mM captan. Ribonuclease H activity was inhibited when AMV reverse transcriptase was preincubated with 1 mM captan before the degradative reaction was initiated. Preincubation of enzyme with polynucleotide before exposure to captan could partially protect the RNase H activity (61 ± 2% activity remained). Deoxyguanosine triphosphate also partially protected the RNase H activity from inhibition by captan (75 ± 9% activity remained). Inhibition of the RNase H activity was completely prevented by preincubating enzyme simultaneously with polynucleotide and dGTP. When separated by glycerol gradients the α subunit and αβ dimer both exhibited RNase H activity, but only the RNase H activity of the α subunit was inhibited by captan. Activity and binding studies revealed that the RNase H and polymerase activities of the α subunit are not susceptible to the interaction of captan when this subunit is in the αβ dimer form. Thus, either β subunit, upon association with a physically blocks the captan binding sites, or the interaction of β subunit with α subunit causes a conformational change in the α subunit making it incapable of binding captan.