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J Biol Chem. 1999 Dec 10;274(50):35768-76.

The motif D loop of human immunodeficiency virus type 1 reverse transcriptase is critical for nucleoside 5'-triphosphate selectivity.

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1
Centre National de la Recherche Scientifique, Ecole Supérieure d'Ingénieurs de Luminy, Architecture et Fonction des Macromolécules Biologiques, 163 avenue de Luminy, 13288 Marseille cedex 9, France.

Abstract

Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the "palm" domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment from Taq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3'-deoxy 3'-azidothymidine 5'-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.

PMID:
10585459
DOI:
10.1074/jbc.274.50.35768
[Indexed for MEDLINE]
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