Examining interactions of HIV-1 reverse transcriptase with single-stranded template nucleotides by nucleoside analog interference

Chandravanu Dash, Timothy S. Fisher, Vinayaka R. Prasad, Stuart F.J. Le Grice

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Crystallographic studies have implicated several residues of the p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase in contacting the single-stranded template overhang immediately ahead of the DNA polymerase catalytic center. This interaction presumably assists in inducing the appropriate geometry on the template base for efficient and accurate incorporation of the incoming dNTP. To investigate this, we introduced nucleoside analogs either individually or in tandem into the DNA template ahead of the catalytic center and investigated whether they induce pausing of the replication machinery before serving as the template base. Analogs included abasic tetrahydrofuran linkages, neutralizing methylphosphonate linkages, and conformationally locked nucleosides. In addition, several Phe-61 mutants were included in our analysis, based on previous data indicating that altering this residue affects both strand displacement synthesis and the fidelity of DNA synthesis. We demonstrate here that altering the topology of the template strand two nucleotides ahead of the catalytic center can interrupt DNA synthesis. Mutating Phe-61 to either Ala or Leu accentuates this defect, whereas replacement with an aromatic residue (Trp) allows the mutant enzyme to bypass the template analogs with relative ease.

Original languageEnglish (US)
Pages (from-to)27873-27881
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number38
DOIs
StatePublished - Sep 22 2006

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Examining interactions of HIV-1 reverse transcriptase with single-stranded template nucleotides by nucleoside analog interference'. Together they form a unique fingerprint.

Cite this