Site-directed mutagenesis of Moloney murine leukemia virus reverse transcriptase. Demonstration of lysine 103 in the nucleotide binding site.
Lys103 and Lys421 of Moloney murine leukemia virus reverse transcriptase have been implicated in the dNTP binding function as judged by their reactivity to a substrate binding site-directed reagent, pyridoxal 5'-phosphate (Basu, A., Nanduri, V. B., Gerard, G. F., and Modak, M. J. (1988) J. Biol. Chem. 263, 1648-1653). To assess the true catalytic importance of the individual lysine residues in Moloney murine leukemia virus reverse transcriptase, we mutated Lys103 and Lys421 to leucine and alanine, respectively. Analysis of the mutant enzymes revealed that mutation at the 103 position had a drastic effect on the DNA polymerase activity whereas the 421 mutation had no effect. Both mutants exhibited normal RNase H activity as well as the ability to bind to RNA or DNA templates as judged by UV-mediated cross-linking of the enzyme to the template primers. The enzyme with mutation at codon 421 (Lys----Ala) exhibited properties that were indistinguishable from the wild type with respect to its mode of catalysis, i.e. preference of template primer and divalent metal ion, RNA- or DNA-dependent DNA polymerase activity, RNase H activity, and the processive mode of DNA synthesis. These observations suggest that only Lys103 and not Lys421 is the catalytically important residue that is involved in the binding of substrate dNTP in Moloney murine leukemia virus reverse transcriptase.
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey/New Jersey Medical School, Newark 07103.
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D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008239:Lysine; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016914:Ribonuclease H; D013698:Templates, Genetic
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H; D008239:Lysine