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J. Biol. Chem., Vol. 277, Issue 46, 43593-43598, November 15, 2002

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Exploring the Effects of Active Site Constraints on HIV-1 Reverse Transcriptase DNA Polymerase Fidelity^*

Janina Cramer, Michael Strerath, Andreas Marx, and Tobias Restle^§

From the  Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Straße 1, 53121 Bonn, Germany and the Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Straße 11, 44227 Dortmund, Germany

To examine the concept of polymerase active site tightness as a criteria for DNA polymerase fidelity, we performed pre-steady-state single nucleotide incorporation kinetic analyses with sugar modified thymidine 5'-triphosphate (TTP) analogues and human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The employed TTP analogues (T^RTP) are modified at the 4'-position of the sugar moiety with alkyl groups, gradually expanding their steric demand. Introduction of a methyl group reduces the maximum rate of nucleotide incorporation by about 200-fold for RT^WT and about 400-fold for RT^M184V. Interestingly, the affinity of RT for the modified nucleotide is only marginally affected. Increasing the size to an ethyl group leads to further reduction of the rate of incorporation and first effects on binding affinities are observed. Finally, substitution for an isopropyl group results not only in a further reduction of incorporation rates but also in a dramatic loss of binding affinity for the nucleotide analogue. By increasing the steric demand the effects on RT^M184V in comparison with RT^WT become progressively more pronounced. Misincorporation of either TTP or T^MeTP opposite a template G causes additional decline in incorporation rates accompanied by a drastic decrease in binding affinities. This results in relative incorporation efficiencies [(k_pol/K_d)_incorrect/(k_pol/K_d)_TTPcorrect] of 4.1 × 10⁵ for TTP and 3.4 × 10⁶ for T^MeTP in case of RT^WT and 1.4 × 10⁵ for TTP and 2.9 × 10⁸ for T^MeTP in case of RT^M184V.

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