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• W2042704478 endingPage "229" @default.
• W2042704478 startingPage "221" @default.
• W2042704478 abstract "Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3′-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent Km values for dNTP. Discrimination between AZTTP (3′-azido-3′-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5–6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G→A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide." @default.
• W2042704478 created "2016-06-24" @default.
• W2042704478 creator A5061687469 @default.
• W2042704478 creator A5064411791 @default.
• W2042704478 date "2005-03-22" @default.
• W2042704478 modified "2023-09-24" @default.
• W2042704478 title "Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114" @default.
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• W2042704478 doi "https://doi.org/10.1042/bj20041056" @default.
• W2042704478 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1134950" @default.
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• W2042704478 hasPublicationYear "2005" @default.
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