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Originally published In Press as doi:10.1074/jbc.M000279200 on March 9, 2000
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J Biol Chem, Vol. 275, Issue 20, 15025-15033, May 19, 2000

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. LathamDagger §, Eva ForgacsDagger , William A. Beard, Rajendra Prasad, Katarzyna Bebenek||, Thomas A. Kunkel||, Samuel H. Wilson, and R. Stephen LloydDagger **

From the Dagger  Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1071 and the  Laboratory of Structural Biology and the || Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

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.

* This work was supported in part by National Institutes of Health Grants ES05355 and ES06766 (to R. S. L.) and National Institutes of Health Grants ES06492 and ES06839 (to S.H.W.) and by grants (to T. A. K. and S. H. W.) from the National Institutes of Health Intramural AIDS Targeted Antiviral Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Current address: Ambion, Inc., 2130 Woodward St., Austin, TX 78744.

** Holder of the Mary Gibbs Jones Distinguished Chair in Environmental Toxicology from the Houston Endowment. To whom correspondence should be addressed. Tel.: 409-772-2179; Fax: 409-772-1790; E-mail: rslloyd@utmb.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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