Metal binding and activation of the ribonuclease H domain from Moloney murine leukemia virus

The RNase H family of enzymes degrades RNA in RNA DNA hybrids in a divalent cation-dependent manner. RNases H from diverse sources such as Escherichia coli and human immunodeficiency virus (HIV) share homologous metal-binding active sites, and the activity of the RNase H domain of reverse transcriptase (RT) is required for retroviral replication. The isolated RNase H domain from HIV RT, however, is inactive. In contrast, the RNase H domain of Moloney murine leukemia virus (MMLV) is active, enabling functional studies. Unlike both E.coli RNase HI and HIV RT, the RNase K activity of MMLV RT shows greater activity in Mn2+ than Mg2+. We investigated the effect of mutations in five conserved active-site residues of the isolated MMLV RNase H domain. Mutations in two carboxylates eliminate metal binding while mutations in other active-site residues allow retention of metal ion affinity. Mutations that inactivate E.coli RNase HI in Mg2+ have similar effects on the Mn2+-dependent activity of MMLV RNase H. These results suggest a similar one-metal catalytic mechanism for the Mn2+- and Mg2+-dependent activities of both prokaryotic and retroviral ribonucleases H.