Probing structure/function relationships of HIV-1 reverse transcriptase with styrene oxide N2-guanine adducts

Eva Forgacs, Gary Latham, William A. Beard, Rajendra Prasad, Katarzyna Bebenek, Thomas A. Kunkel, Samuel H. Wilson, R. Stephen Lloyd

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

Details of the interactions between the human immunodeficiency virus (HIV-1) reverse transcriptase and substrate DNA were probed both by introducing site-specific and stereospecific modifications into DNA and by altering the structure of potential critical residues in the polymerase. Unadducted 11-mer DNAs and 11-mer DNAs containing R and S enantiomers of styrene oxide at N2-guanine were ligated with two additional oligonucleotides to create 63-mers that served as templates for HIV-1 reverse transcriptase replication. Oligonucleotides that primed synthesis 5 bases 3' to the adducts could be extended up to 1 base 3' and opposite the lesion. However, when the positions of the 3'-OH of the priming oligonucleotides were placed 1, 2, 3, 4, 5, and 6 bases downstream of the styrene oxide guanine adducts, replication was initiated, only to be blocked after incorporating 4, 5, 6, and 7 bases beyond the lesion. The sites of this adduct-induced termination corresponded to the position of the DNA where α-helix H makes contact with the DNA minor groove, 3-5 bases upstream of the growing 3' end. In addition, mutants of the polymerase in α-helix H (W266A and G262A) alter the termination probabilities caused by these DNA adducts, suggesting that α-helix H is a sensitive monitor of modifications in the minor groove of newly synthesized template-primer DNA several bases distal to the 3'-OH.

Original languageEnglish (US)
Pages (from-to)8525-8530
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number13
DOIs
StatePublished - Mar 28 1997

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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