Exploring the effects of active site constraints on HIV-1 reverse transcriptase DNA polymerase fidelity.

Abstract:

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(R)TP) 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(Me)TP 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 x 10(-5) for TTP and 3.4 x 10(-6) for T(Me)TP in case of RT(WT) and 1.4 x 10(-5) for TTP and 2.9 x 10(-8) for T(Me)TP in case of RT(M184V).

Polymerases:

Topics:

Mutational Analysis, Health/Disease, Nucleotide Analogs / Template Lesions, Structure and Structure/Function, Nucleotide Incorporation, Reverse Transcriptase

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
HIV RT Cramer J2002 Reverse Transcriptase Activity Yes
HIV RT Cramer J2002 Associated condition HIV
HIV RT M184V Cramer J2002 Reverse Transcriptase Activity Yes
HIV RT M184V Cramer J2002 Associated condition HIV

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