Isolation of cloned moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity

Abstract

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. coll, 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-HLV reverse transcriptase reside within separate structural domains. © 1988 IRL Press Limited, Oxford, England.