Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity.
Retroviral reverse transcriptase possesses DNA polymerase and ribonuclease H (RNase H) activity within a single polypeptide. Chemical or proteolytic treatment of reverse transcriptase has been used in the past to produce enzyme that is missing DNA polymerase activity and retains RNase H activity. It has not been possible to obtain reverse transcriptase that lacks RNase H but retains DNA polymerase activity. We have constructed a novel deletion derivative of the cloned Moloney murine leukemia virus (M-MLV) reverse transcriptase gene, expressed the gene in E. coli, and purified the protein to near homogeneity. The purified enzyme has a fully active DNA polymerase, but has no detectable RNase H activity. These results are consistent with, but do not prove, the conclusion that the DNA polymerase and RNase H activities of M-MLV reverse transcriptase reside within separate structural domains.
D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D005796:Genes; D005814:Genes, Viral; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*
D012194:RNA-Directed DNA Polymerase; D012260:Ribonucleases; D004262:DNA Restriction Enzymes