K65R and K65A Substitutions in HIV-1 Reverse Transcriptase Enhance Polymerase Fidelity by Decreasing Both dNTP Misinsertion and Mispaired Primer Extension Efficiencies

Scott J. Garforth, Robert A. Domaoal, Chisanga Lwatula, Mark J. Landau, Amanda J. Meyer, Karen S. Anderson, Vinayaka R. Prasad

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Lys65 residue, in the fingers domain of human immunodeficiency virus reverse transcriptase (RT), interacts with incoming dNTP in a sequence-independent fashion. We showed previously that a 5-amino-acid deletion spanning Lys65 and a K65A substitution both enhanced the fidelity of dNTP insertion. We hypothesized that the Lys65 residue enhances dNTP misinsertion via interactions with the λ -phosphate of the incoming dNTP. We now examine this hypothesis in pre-steady-state kinetic studies using wild-type human immunodeficiency virus-1 RT and two substitution mutants, K65A and K65R. K65R mutation did not greatly increase misinsertion fidelity, but K65A mutation led to higher incorporation fidelity. For a misinsertion to become a permanent error, it needs to be accompanied by the extension of the mispaired terminus thus formed. Both mutants and the wild-type enzyme discriminated against the mismatched primer at the catalytic step (kpol). Additionally, K65A and K65R mutants displayed a further decrease in mismatch extension efficiency, primarily at the level of dNTP binding. We employed hydroxyl radical footprinting to determine the position of the RT on the primer/template. The wild-type and Lys65-substituted enzymes occupied the same position at the primer terminus; the presence of a mismatched primer terminus caused all three enzymes to be displaced to a -2 position relative to the primer 3' end. In the context of an efficiently extended mismatched terminus, the presence of the next complementary nucleotide overcame the displacement, resulting in a complex resembling the matched terminus. The results are consistent with the observed reduction in kpol in mispaired primer extension being due to the position of the enzyme at a mismatched terminus. Our work shows the influence of the stabilizing interactions of Lys65 with the incoming dNTP on two different aspects of polymerase fidelity.

Original languageEnglish (US)
Pages (from-to)33-44
Number of pages12
JournalJournal of Molecular Biology
Volume401
Issue number1
DOIs
StatePublished - Aug 2010

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Enzymes
HIV Reverse Transcriptase
Mutation
RNA-Directed DNA Polymerase
Hydroxyl Radical
Fingers
Nucleotides
Phosphates
Amino Acids
Human immunodeficiency virus 1 reverse transcriptase

Keywords

  • HIV-1 reverse transcriptase
  • NRTI resistance mutation
  • Polymerase fidelity
  • Pre-steady-state kinetics of HIV-1 RT
  • Site-specific footprinting

ASJC Scopus subject areas

  • Molecular Biology

Cite this

K65R and K65A Substitutions in HIV-1 Reverse Transcriptase Enhance Polymerase Fidelity by Decreasing Both dNTP Misinsertion and Mispaired Primer Extension Efficiencies. / Garforth, Scott J.; Domaoal, Robert A.; Lwatula, Chisanga; Landau, Mark J.; Meyer, Amanda J.; Anderson, Karen S.; Prasad, Vinayaka R.

In: Journal of Molecular Biology, Vol. 401, No. 1, 08.2010, p. 33-44.

Research output: Contribution to journalArticle

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abstract = "Lys65 residue, in the fingers domain of human immunodeficiency virus reverse transcriptase (RT), interacts with incoming dNTP in a sequence-independent fashion. We showed previously that a 5-amino-acid deletion spanning Lys65 and a K65A substitution both enhanced the fidelity of dNTP insertion. We hypothesized that the Lys65 residue enhances dNTP misinsertion via interactions with the λ -phosphate of the incoming dNTP. We now examine this hypothesis in pre-steady-state kinetic studies using wild-type human immunodeficiency virus-1 RT and two substitution mutants, K65A and K65R. K65R mutation did not greatly increase misinsertion fidelity, but K65A mutation led to higher incorporation fidelity. For a misinsertion to become a permanent error, it needs to be accompanied by the extension of the mispaired terminus thus formed. Both mutants and the wild-type enzyme discriminated against the mismatched primer at the catalytic step (kpol). Additionally, K65A and K65R mutants displayed a further decrease in mismatch extension efficiency, primarily at the level of dNTP binding. We employed hydroxyl radical footprinting to determine the position of the RT on the primer/template. The wild-type and Lys65-substituted enzymes occupied the same position at the primer terminus; the presence of a mismatched primer terminus caused all three enzymes to be displaced to a -2 position relative to the primer 3' end. In the context of an efficiently extended mismatched terminus, the presence of the next complementary nucleotide overcame the displacement, resulting in a complex resembling the matched terminus. The results are consistent with the observed reduction in kpol in mispaired primer extension being due to the position of the enzyme at a mismatched terminus. Our work shows the influence of the stabilizing interactions of Lys65 with the incoming dNTP on two different aspects of polymerase fidelity.",
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AU - Garforth, Scott J.

AU - Domaoal, Robert A.

AU - Lwatula, Chisanga

AU - Landau, Mark J.

AU - Meyer, Amanda J.

AU - Anderson, Karen S.

AU - Prasad, Vinayaka R.

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