Vertical-scanning Mutagenesis of a Critical Tryptophan in the “Minor Groove Binding Track” of HIV-1 Reverse Transcriptase

Abstract

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 Gly 262 and Trp 266 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 Trp 266 . 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 Trp 266 mutants and an alanine mutant of Gly 262 terminated synthesis on styrene oxideadducted 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.Rapid and efficient DNA polymerization requires stable interactions between the polymerase and the template-primer to facilitate highly processive DNA synthesis. When examined alone, the leading/lagging strand polymerases from human, Escherichia coli, and T4 bacteriophage are poorly processive, but become highly processive after binding accessory proteins (1, 2). Each of these replication machines also exhibits high fidelity. Although the presence of an editing 3Ј 3 5Ј exonuclease activity contributes significantly to this low error rate, evidence supports a general correlation between polymerase processivity and fidelity (3).Human immunodeficiency virus type 1 (HIV-1) 1 reverse transcriptase (RT) is a heterodimer of p66 and p51 subunits and exhibits both polymerase and RNase H activities. However, RT does not have an associated 3Ј 3 5Ј exonuclease function (4) and is only moderately processive (5). RT is one of the most error-prone DNA polymerases examined to date, averaging one error per 2 kilobase pairs replicated (4, 6). RT appears to lack a true processivity factor in vivo, although HIV nucleocapsid protein may operate in a manner similar to single-stranded binding protein to increase RT processivity through regions of DNA secondary structure (7). Since the rapid mutation rate of HIV is widely considered a major stumbling block in the development of therapies to combat acquired immunodeficiency syndro...

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