Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus.
Two RNase H (RNA-DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) activities separable by Sephadex G-100 gel filtration were identified in lysates of Moloney murine sarcoma-leukemia virus (MSV). The larger enzyme, which we have called RNase H-I, represented about 10% of the RNase H activity in the virion. RNase H-I (i) copurified with RNA-directed DNA polymerase from the virus, (ii) had a sedimentation coefficient of 4.4S (corresponds to an apparent mol wt of 70,000), (iii) required Mn-2+ (2 mM optimum) for activity with a [3-h]poly(A)-poly(dT) substrate, (iv) eluted from phosphocellulose at 0.2 M KC1, and (v) degraded [3-H]poly(A)-poly(dT) and [3-H]poly(C)-poly(dG) at approximately equal rates. The smaller enzyme, designated RNase H-II, which represented the majority of the RNase H activity in the virus preparation, was shown to be different since it (i) had no detectable, associated DNA polymerase activity, (ii) had a sedmimentation coefficient of 2.6S (corresponds to an apparent mol wt of 30,000), (iii) preferred Mg-2+ (10 to 15 mM optimum) over Mn-2+ (5 to 10 mM optimum) 2.5-fold for the degradation of [3-H]poly(A)-poly(dT), and (iv) degraded [3-H]poly(A)-poly(dT) 6 and 60 times faster than [3-H]poly(C)-poly(dG) in the presence of Mn-2+ and Mg-2+, respectively. Moloney MSV DNA polymerase (RNase H-I), purified by Sephadex G-100 gel filtration followed by phosphocellulose, poly(A)-oligo(dT)-cellulose, and DEAE-cellulose chromatography, transcribed heteropolymeric regions of avian myeloblastosis virus 70S RNA at a rate comparable to avian myeloblastosis virus DNA polymerase purified by the same procedure.
D001354:Avian Leukosis Virus / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002850:Chromatography, Gel; D005816:Genetic Complementation Test; D008274:Magnesium; D008345:Manganese; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009693:Nucleic Acid Hybridization; D011119:Polynucleotides; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification; D012260:Ribonucleases / Q000302:isolation & purification; D014158:Transcription, Genetic; D014316:Tritium
D011119:Polynucleotides; D014316:Tritium; D008345:Manganese; D012194:RNA-Directed DNA Polymerase; D012260:Ribonucleases; D008274:Magnesium