Vertical-scanning mutagenesis of a critical tryptophan in the 'minor groove binding track' of HIV-1 reverse transcriptase. Major groove DNA adducts identify specific protein interactions in the minor groove

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

Research output: Contribution to journalArticle

30 Scopus citations


Biochemical and molecular modeling studies of human immunodeficiency virus type 1 reverse transcriptase (RT) have revealed that a structural element, the minor groove binding track (MGBT), is important for both replication frameshift fidelity and processivity. The MGBT interactions occur in the DNA minor groove from the second through sixth base pair from the primer 3'-terminus where the DNA undergoes a structural transition from A- like to B-form DNA. Alanine-scanning mutagenesis had previously demonstrated that Gly262 and Trp266 of the MGBT contributes important DNA interactions. To probe the molecular interactions occurring in this critical region, eight mutants of RT were studied in which alternate residues were substituted for Trp266. These enzymes were characterized in primer extension assays in which the template DNA was adducted at a single adenine by either R- or S-enantiomers of styrene oxide. These lesions failed to block DNA polymerization by wild-type RT, yet the Trp266 mutants and an alanine mutant of Gly262 terminated synthesis on styrene oxide-adducted templates. Significantly, the sites of termination occurred primarily 1 and 3 bases following adduct bypass, when the lesion was positioned in the major groove of the template-primer stem. These results indicate that residue 266 serves as a 'protein sensor' of altered minor groove interactions and identifies which base pair interactions are altered by these lesions. In addition, the major groove lesion must alter important structural transitions in the template-primer stem, such as minor groove widening, that allow RT access to the minor groove.

Original languageEnglish (US)
Pages (from-to)15025-15033
Number of pages9
JournalJournal of Biological Chemistry
Issue number20
StatePublished - May 19 2000


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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