Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H.
DNA polymerase was purified from a cloned isolate of Moloney murine leukemia virus (M-MuLV). Purified M-MuLV DNA polymerase, upon analysis by polyacrylamide gel electrophoresis, showed one major polypeptide of mol wt 80,000. Estimation of molecular weight from the sedimentation rate of the purifed enzyme in a glycerol gradient was consistent with a structure containing one polypeptide. M-MuLV DNA polymerase could transcribe ribopolymers, deoxyribopolymers, and heteropolymers as efficiently as did purified DNA polymerase from avian myeloblastosis virus (AMV). M-MuLV DNA polymerase, however, transcribed native 70S viral RNA less efficiently than did AMV DNA polymerase. Addition of oligo(dT) enhanced five to tenfold the transcription of 70S viral RNA by M-MuLV DNA polymerase. Purified enzyme also exhibited nuclease activity (RNase H) that selectively degraded the RNA moiety of the RNA-DNA hybrid. It did not degrade single-stranded RNA, single-stranded DNA, double-stranded RNA, and double-stranded DNA. M-MuLV DNA polymerase-associated RNase H acted as a random exonuclease. When [3-H]poly(A)-poly(dT) was used as a substrate, the size of the M-MuLV DNA polymerase-associated RHase H digested product was larger than the size of the digestion products by AMV DNA polymerase. The oligonucleotide digestion products could be further digested to 5'-AMP by snake venom phosphodiesterase, indicating that the products were terminated by 3'-OH groups. Alkaline hydrolysis of the oligonucleotide digestion products generated pAp, suggesting that M-MuLV DNA polymerase-associated RNase H cleaves at the 3' side of the 3',5'-phosphodiester bond. The ratios of the rates of DNA polymerase activity and RNase H activity were not significantly different in the murine and avian enzymes.
D002501:Centrifugation, Zonal; D004591:Electrophoresis, Polyacrylamide Gel; D008274:Magnesium; D008345:Manganese; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009693:Nucleic Acid Hybridization; D010455:Peptides / Q000032:analysis; D010727:Phosphoric Diester Hydrolases; D011119:Polynucleotides; D012194:RNA-Directed DNA Polymerase / Q000032:analysis / Q000302:isolation & purification; D012260:Ribonucleases / Q000032:analysis / Q000302:isolation & purification; D013698:Templates, Genetic; D013942:Thymine Nucleotides; D014158:Transcription, Genetic; D014316:Tritium
D010455:Peptides; D011119:Polynucleotides; D013942:Thymine Nucleotides; D014316:Tritium; D008345:Manganese; D012194:RNA-Directed DNA Polymerase; D012260:Ribonucleases; D010727:Phosphoric Diester Hydrolases; D008274:Magnesium