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 H activity of MMLV RT shows greater activity in Mn(2+) than Mg(2+). 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 Mg(2+) have similar effects on the Mn(2+)-dependent activity of MMLV RNase H. These results suggest a similar one-metal catalytic mechanism for the Mn(2+)- and Mg(2+)-dependent activities of both prokaryotic and retroviral ribonucleases H.