Mechanistic independence of avian myeloblastosis virus DNA polymerase and ribonuclease H.
Differential inhibition conditions were established for the DNA polymerase and RNase H activities of avian myeloblastosis virus (AMV) with ether-disrupted AMV and a purified enzyme preparation. The RNase H activity of ether-disrupted AMV with (rA)(n).(dT)(n) and (rA)(n).(dT)(11) as substrates was inhibited 80 to 100% by preincubation with NaF at a final reaction concentration of 27 to 30 mM. Under these conditions, the DNA polymerase activity was inhibited only 0 to 20%. Similar inhibitions were found with exogenous Rous sarcoma virus 35S and 70S RNA.DNA hybrid and phiX174 DNA.RNA hybrid as substrates. Studies were also performed with a purified enzyme preparation, in which the two activities essentially co-purified. The RNase H activity was inhibited >80% by 150 mM KCl with three different hybrid substrates, whereas the DNA polymerase activity was uninhibited. The DNA polymerase was completely inactivated by heat denaturation at 41 C or by omission of the deoxytriphosphates from the reaction mixture; the RNase H remained active. These differential inhibition conditions were used to compare the size of the DNA product synthesized with and without simultaneous RNase H action and to examine the effect of inhibition of the DNA polymerase on the size of the RNase H products. The size of the products of one activity was not affected by inhibition of the other activity. These results suggest that the AMV DNA polymerase and RNase H are not coupled mechanistically.
D001354:Avian Leukosis Virus / Q000201:enzymology*; D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D004254:DNA Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005459:Fluorides / Q000494:pharmacology; D006358:Hot Temperature; D009693:Nucleic Acid Hybridization; D011189:Potassium Chloride / Q000494:pharmacology; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D014316:Tritium
D014316:Tritium; D011189:Potassium Chloride; D012964:Sodium; D004254:DNA Nucleotidyltransferases; D012260:Ribonucleases; D005459:Fluorides