title: 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.
abstract: 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.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Konishi
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Hisayoshi
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Kanta
authors.initials: K
authors.lastname: Yokokawa
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/Nicolás Cabrera 1, Campus de Cantoblanco, 28049 Madrid, Spain.
authors.forename: Verónica
authors.initials: V
authors.lastname: Barrioluengo
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/Nicolás Cabrera 1, Campus de Cantoblanco, 28049 Madrid, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Menéndez-Arias
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Electronic address: yasukawa@kais.kyoto-u.ac.jp.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008979:Moloney murine leukemia virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D055550:Protein Stability; D012313:RNA / Q000032:analysis / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H
keywords: Moloney murine leukemia virus; RNase H activity; Reverse transcriptase; Template–primer; Thermostabilization