Residues in the alphaH and alphaI helices of the HIV-1 reverse transcriptase thumb subdomain required for the specificity of RNase H-catalyzed removal of the polypurine tract primer.

Important note: Information in this article was accurate in 1999. The state of the art may have changed since the publication date.

Residues in the alphaH and alphaI helices of the HIV-1 reverse transcriptase thumb subdomain required for the specificity of RNase H-catalyzed removal of the polypurine tract primer.

J Biol Chem. 1999 Jul 9;274(28):19885-93. Unique Identifier : AIDSLINE MED/99321920
Powell MD; Beard WA; Bebenek K; Howard KJ; Le Grice SF; Darden TA; Kunkel TA; Wilson SH; Levin JG; Laboratory of Molecular Genetics, NICHD, National Institutes of; Health, Bethesda, Maryland 20892, USA.

Abstract: During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the alphaH and alphaI helices in the thumb subdomain play in specific RNase H cleavage at the 3'-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3'-end. Further analysis of alphaH mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.

Keywords: JOURNAL ARTICLE Alanine/GENETICS Base Sequence DNA/BIOSYNTHESIS Human HIV-1/*ENZYMOLOGY HIV-1 Reverse Transcriptase/*CHEMISTRY/GENETICS Kinetics Models, Molecular Molecular Sequence Data Mutation Nucleic Acid Conformation *Protein Structure, Secondary Ribonuclease H, Calf Thymus/*METABOLISM RNA/*METABOLISM Substrate Specificity Support, U.S. Gov't, P.H.S. Tryptophan/GENETICS
991030
A99A0879

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