Human immunodeficiency virus type 1 reverse transcriptase: role of Tyr115 in deoxynucleotide binding and misinsertion fidelity of DNA synthesis.
Tyr115 is located in the vicinity of the polymerase catalytic site of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Site-directed mutagenesis was used to generate variant enzymes having Phe, Trp, Ala, Ser, Asp or Lys instead of Tyr115. The substitution of Tyr115 by Phe renders a fully active polymerase, displaying similar kinetic parameters, processivity and misinsertion fidelity of DNA synthesis as the wild-type enzyme. In contrast, the replacement of Tyr by Asp or Lys produced enzymes with a very low polymerase activity. The activity of the variant enzymes having Trp, Ala or Ser instead of Tyr115 was reduced significantly, particularly when poly(rA)484 was used as template. This effect was caused by a dramatic increase in the Km value for dTTP, and was detected using a DNA template mimicking a proviral HIV-1 gag sequence. Misinsertion fidelity assays revealed that mutants Y115W, Y115A and Y115S had a higher misinsertion efficiency than the wild-type reverse transcriptase. The low fidelity of these mutants appears to be related to nucleotide recognition rather than altered DNA-DNA template-primer interactions. The effects observed on the steady state kinetic constants, processivity and fidelity were mediated by the 66 kDa subunit, as demonstrated using chimeric heterodimers with the Y115A substitution in either p66 or p51.
Martín-Hernández
A M
AM
Centro de Biología Molecular Severo Ochoa, CSIC-Universidad Autónoma de Madrid, Spain.
Domingo
E
E
Menéndez-Arias
L
L
D004261:DNA Replication; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D014443:Tyrosine / Q000737:chemistry*
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D054303:HIV Reverse Transcriptase