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.
Blain
S W
SW
Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA.
Goff
S P
SP
D000595:Amino Acid Sequence; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008345:Manganese; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D012259:Ribonuclease, Pancreatic; D008274:Magnesium