Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H

J Virol. 1975 Apr;15(4):843-54. doi: 10.1128/JVI.15.4.843-854.1975.

Abstract

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.

MeSH terms

  • Centrifugation, Zonal
  • Electrophoresis, Polyacrylamide Gel
  • Magnesium
  • Manganese
  • Molecular Weight
  • Moloney murine leukemia virus / enzymology*
  • Nucleic Acid Hybridization
  • Peptides / analysis
  • Phosphoric Diester Hydrolases
  • Polynucleotides
  • RNA-Directed DNA Polymerase* / analysis
  • RNA-Directed DNA Polymerase* / isolation & purification
  • Ribonucleases* / analysis
  • Ribonucleases* / isolation & purification
  • Templates, Genetic
  • Thymine Nucleotides
  • Transcription, Genetic
  • Tritium

Substances

  • Peptides
  • Polynucleotides
  • Thymine Nucleotides
  • Tritium
  • Manganese
  • RNA-Directed DNA Polymerase
  • Ribonucleases
  • Phosphoric Diester Hydrolases
  • Magnesium