Differential effects of moloney murine leukemia virus reverse transcriptase mutations on RNase H activity in Mg2+ and Mn2+

We have previously described the in vitro and in vivo characterization of a panel of mutations affecting the RNase H domain of Moloney murine leukemia virus reverse transcriptase (Blain, S. W., and Goff, S. P. (1993) J. Biol, Chem, 268, 23585-23592; Blain, S. W., and Goff, S. P. (1995) J. Virol, 69, 4440-4452), We were intrigued by a discrepancy between in vitro and in vivo RNase H results for two of the mutants, While Delta C and Delta 5E appeared to have nearly wild-type RNase H activity in vitro, they were unable to degrade their genomic RNA in vivo and thus were effectively RNase H null mutants in this context, In this present report, we describe the differential effects of these mutations on RNase H activity in vitro in the presence of Mg2+ versus Mn2+: mutants Delta C and Delta 5E were active in the presence of the less biologically relevant Mn2+ and not in the presence of Mg2+. We also describe three mutants with only partial activity in Mg2+, The presence of the different cations can also affect DNA polymerization and processivity of an RNase H-deficient mutant.