title: Mechanistic insights into the role of Val75 of HIV-1 reverse transcriptase in misinsertion and mispair extension fidelity of DNA synthesis.
abstract: The side chain of Val75 stabilizes the fingers subdomain of the human immunodeficiency virus type 1 reverse transcriptase (RT), while its peptide backbone interacts with the single-stranded DNA template (at nucleotide +1) and with the peptide backbone of Gln151. Specific DNA polymerase activities of mutant RTs bearing amino acid substitutions at position 75 (i.e., V75A, V75F, V75I, V75L, V75M, V75S and V75T) were relatively high. Primer extension experiments carried out in the absence of one deoxyribonucleoside-triphosphate suggested that mutations did not affect the accuracy of the RT, except for V75A, V75F, V75I, and to a lesser extent V75T. The fidelity of RTs bearing mutations V75F and V75I increased 1.8- and 3-fold, respectively, as measured by the M13 lacZ alpha forward mutation assay, while V75A showed 1.4-fold decreased accuracy. Steady- and pre-steady-state kinetics demonstrated that the increased fidelity of V75I and V75F was related to their decreased ability to extend mismatched template-primers, while misincorporation efficiencies were not significantly affected by mutations. The increased mispair extension fidelity of mutant V75I RT could be attributed to the nucleotide affinity loss, observed in reactions with mismatched template-primers. Altogether, these data suggest that Val75 interactions with the 5' template overhang are important determinants of fidelity.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28049 Madrid, Spain.
authors.forename: Tania
authors.initials: T
authors.lastname: Matamoros
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
authors.forename: Luis
authors.initials: L
authors.lastname: Menéndez-Arias
mesh_terms: D019943:Amino Acid Substitution; D017104:Bacteriophage M13 / Q000235:genetics; D020137:Base Pair Mismatch; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013698:Templates, Genetic; D014633:Valine / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase; D014633:Valine