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

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 [14C]captan bound to the alpha subunit in a ratio of 10:1 and to a 32,000 d polypeptide in a ratio of 4:1. Neither the alpha beta nor the beta subunit bound [14C]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 alpha subunit and alpha beta dimer both exhibited RNase H activity, but only the RNase H activity of the alpha subunit was inhibited by captan. Activity and binding studies revealed that the RNase H and polymerase activities of the alpha subunit are not susceptible to the interaction of captan when this subunit is in the alpha beta dimer form.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of captan to DNA polymerase I from Escherichia coli and the concomitant effect on 5'----3' exonuclease activity.

Captan (N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide) was shown to bind to DNA polymerase I from Escherichia coli. The ratio of [14C] captan bound to DNA pol I was 1:1 as measured by filter binding studies and sucrose gradient analysis. Preincubation of enzyme with polynucleotide prevented the binding of captan, but preincubation of enzyme with dGTP did not. Conversely, when the enzyme was preincubated with captan, neither polynucleotide nor dGTP binding was blocked. The modification of the enzyme by captan was described by an irreversible second-order rate process with a rate of 68 +/- 0.7 M-1 s-1. The interaction of captan with DNA pol I altered each of the three catalytic functions. The 3'----5' exonuclease and polymerase activities were inhibited, and the 5'----3' exonuclease activity was enhanced. In order to study the 5'----3' exonuclease activity more closely, [3H]hpBR322 (DNA-[3H]RNA hybrid) was prepared from pBR322 plasmid DNA and used as a specific substrate for 5'----3' exonuclease activity. When either DNA pol I or polynucleotide was preincubated with 100 microM captan, 5'----3' exonuclease activity exhibited a doubling of reaction rate as compared to the untreated sample. When 100 microM captan was added to the reaction in progress, 5'----3' exonuclease activity was enhanced to 150% of the control value. Collectively, these data support the hypothesis that captan acts on DNA pol I by irreversibly binding in the template-primer binding site associated with polymerase and 3'----5' exonuclease activities. It is also shown that the chemical reaction between DNA pol I and a single captan molecule proceeds through a Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of the exonuclease activities of DNA polymerase I by captan.

DNA polymerase I is a multifaceted enzyme with one polymerizing and two exonuclease activities. Captan was previously shown to be an inhibitor of this enzyme's polymerizing activity and this report measures the effects of captan on the two exonuclease activities. When the holoenzyme was tested, captan enhanced the degradation of poly(dA-dT), T7 DNA and, to a significantly lesser extent, heat-denatured DNA. However, when the effects of captan were tested as a function of substrate concentration, the stimulatory influence was measured only at high substrate concentrations. At low concentrations of DNA, captan was inhibitory. Inhibition and enhancement each showed an ED50 of the same value (approx. 100 microM). By assaying the two exonuclease activities separately it was shown that the differential effect on the holoenzyme by captan was the result of a combined inhibition of the 3'----5' exonuclease and enhancement of the 5'----3' exonuclease. Klenow fragment with poly(dA-dT) as substrate was used to assay for 3'----5' exonuclease activity. Captan inhibited this exonuclease and the inhibition could be prevented by the addition of greater concentrations of substrate. Holoenzyme and poly(rA)-poly(dT) were used to assay for 5'----3' exonucleolysis, which was enhanced at higher concentrations of substrate in the presence of captan.

Differential effects of captan on DNA polymerase and ribonuclease H activities of avian myeloblastosis virus reverse transcriptase.

Captan was used as an inhibitor of avian myeloblastosis virus reverse transcriptase to study the polymerase and RNase H catalytic activities. With purified enzyme, RNase H activity was 10-fold more sensitive to captan than was either the DNA-dependent or RNA-dependent DNA polymerase activity. Inhibition of the RNA-dependent polymerase activity could be prevented by dTTP. Conversely, inhibition of this polymerase activity was enhanced by template/primer. The calculated KdTTP of the uninhibited reaction was 5.6 microM. Kinetic studies allow for the proposition of a model for the interaction of captan with the polymerase active center. RNase H activity showed a sigmoidal relationship between activity and substrate concentration. Nuclease activity decreased in Vmax with no change in the Hill coefficient in the presence of captan. Addition of dithiothreitol to the incubation cocktail prevented inhibition by captan of both RNA-dependent polymerase and RNase H activities, suggesting that the (trichloromethyl)thio moiety of captan is involved in the inhibitory action. Captan inhibition suggests the presence of essential amino residues in both polymerase and RNase H active centers.