Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I).
The mechanism of action of the ribonuclease H (RNase H) activity associated with Moloney murine leukemia virus RNA-directed DNA polymerase (RNase H I) and the two-subunit (alpha beta) form of avian myeloblastosis virus DNA polymerase were compared by utilizing the model substrate (A)n.(dT)n and polyacrylamide gel electrophoresis in 7 M urea to analyze digestion products. Examination on 25% polyacrylamide gels revealed that a larger proportion of the RNase H I oligonucleotide products generated by limited digestion of [3H](A)(1100).(dT)n were acid insoluble (15-26 nucleotides long) than acid soluble (less than 15 nucleotides long), while the opposite was true for products generated by alpha beta RNase H. RNase H I was capable of attacking RNA in RNA.DNA in the 5' to 3' and 3' to 5' directions, as demonstrated by the use of [3H,3'- or 5'-32P](A)(380).(dT)n and cellulose--[3H](A)n.(dT)n. Both RNase H I and alpha beta RNase H degraded [3H]-(A)n.(dT)n with a partially processive mechanism, based upon classical substrate competition experiments and analyses of the kinetics of degradation of [3H,3'- or 5'-32P](A)(380).(dT)n. That is, both enzymes remain bound to a RNA.DNA substrate through a finite number of hydrolytic events but dissociate before the RNA is completely degraded. Both RNase H I and alpha beta RNase H were capable of degrading [14C](A)n in [3H](C)n-[14C](A)n-[32P](dA)n.(dT)n, suggesting that retroviral RNase H is capable of removing the tRNA primer at the 5' terminus of minus strand DNA at the appropriate time during retroviral DNA synthesis in vitro.
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D002250:Carbon Radioisotopes; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D010761:Phosphorus Radioisotopes; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D016914:Ribonuclease H; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium
D002250:Carbon Radioisotopes; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D014316:Tritium; D012194:RNA-Directed DNA Polymerase; D004720:Endonucleases; D012260:Ribonucleases; D016914:Ribonuclease H