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Protein Engineering, Vol. 12, No. 11, 975-980, November 1999
© 1999 Oxford University Press

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

Eric R. Goedken and Susan Marqusee1

Department of Molecular and Cell Biology, University of California, Berkeley, 229 Stanley Hall, Berkeley, CA 94720, USA

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 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.

Keywords: divalent metals/nucleic acid hydrolysis/retrovirus/RNase H/reverse transcriptase

1 To whom correspondence should be addressed;email: marqusee{at}zebra.berkeley.edu


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