Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation

•MMLV RT mutants (E286R, E302K, and L435R) have higher thermostability than wild-type enzyme (WT).•WT and all mutants had similar affinities for heteropolymeric DNA/DNA and RNA/DNA complexes.•Unlike WT, all mutants were devoid of RNase H activity.•It is proposed that these three mutations increase the stability by abolishing the RNase H activity.

We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template–primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by which those mutations increase the thermal stability of the RT. The three single-mutants (E286R, E302K, and L435R), an RNase H-deficient MMLV RT (carrying the RNase H-inactivating mutation D524A), a quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) and the wild-type enzyme (WT) were produced in Escherichia coli. All RTs exhibited similar dissociation constants (Kd) for heteropolymeric DNA/DNA (2.9–6.5 nM) and RNA/DNA complexes (1.2–2.9 nM). Unlike the WT, mutant enzymes (E286R, E302K, L435R, D524A, and MM4) were devoid of RNase H activity, and were not able to degrade RNA in RNA/DNA complexes. These results suggest that the mutations, E286R, E302K, and L435R increase the thermostability of MMLV RT not by increasing its affinity for T/P but by abolishing its RNase H activity.