Role of methionine 184 of human immunodeficiency virus type-1 reverse transcriptase in the polymerase function and fidelity of DNA synthesis.
Methionine 184 of HIV-1 RT is a constituent of the catalytically crucial and highly conserved YXDD motif in the reverse transcriptase class of enzymes. We investigated the role of this residue by substituting it with Ala and Val by site-directed mutagenesis followed by extensive characterization of the two mutant enzymes. The kinetic parameters governing DNA synthesis directed by RNA and DNA templates indicated that both M184A and M184V mutants are catalytically as efficient as the wild type enzyme. Photoaffinity labeling of both the mutant and the wild type enzyme exhibited an identical affinity for RNA-DNA and DNA-DNA template primers. We further demonstrate that M-->V substitution at 184 position significantly increases the fidelity of DNA synthesis while M-->A substitution results in a highly error-prone enzyme without having compromised its efficiency of DNA synthesis. The M184V mutant exhibited a 25-45-fold increase in mismatch selectivity (ratio of k(cat)/K(m) of correct versus incorrect nucleotides) as compared to the WT enzyme. This pattern of error-prone synthesis is also confirmed by examining the abilities of the enzyme-(template-primer) covalent complexes to incorporate correct versus incorrect nucleotide onto the immobilized template-primer. The nature of error-prone synthesis by the M184A mutant shows an increase in both the mismatch synthesis and extension of the mismatched primer termini. Using a three-dimensional molecular model of the ternary complex of HIV-1 RT, template-primer, and dNTP, we observe that the strategic location of M184 may allow it to interact with the sugar moiety of either the primer nucleotide or the dNTP substrate.
Pandey
V N
VN
Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School, Newark 07103, USA.
Kaushik
N
N
Rege
N
N
Sarafianos
S G
SG
Yadav
P N
PN
Modak
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009692:Nucleic Acid Heteroduplexes; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D008715:Methionine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase