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Journal of Molecular Evolution

, Volume 47, Issue 1, pp 73–80 | Cite as

Functional Analysis of HIV-1 Reverse Transcriptase Motif C: Site-Directed Mutagenesis and Metal Cation Interaction

  • Verónica  Valverde-Garduño
  • Patricio  Gariglio
  • Lourdes  Gutiérrez

Abstract.

Motif C, present in all polymerases, has been proposed to be part of the catalytic and metal binding site of the enzyme, suggesting that polymerases have a common origin. Previously, we have shown that the metal ion manganese induces alterations in nucleotide substrate specificity in some polymerases. However, it is not known if the active site responsible for incorporation of nonspecific substrates is the same as that which incorporates specific ones. Here we show that manganese enables HIV-1 reverse transcriptase (RT) to incorporate rNTP's using RNA as a template, thus behaving as an RNA replicase. Also, we show that the mutation D186H in motif C strongly affects the natural DNA polymerase activity and that the RNA replicase activity becomes undetectable, suggesting that both activities depend on the same active site. This mutation changes the metal ion preference, with mutant RT presenting only 0.5% of the wild-type DNA polymerase activity in the presence of magnesium but 1.6% of the same activity in the presence of manganese. This variation in cation preference suggests that residue D186 is part of the metal binding site. Since residue D186 of motif C is essential for both activities and appears to be involved in the binding of an important cation needed for the specific activity, our results support the idea of a common origin for all polymerases, from an ancestral unspecified polymerase containing at least motif C.

Key words: Polymerases — Reverse transcriptase — Specificity 

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Copyright information

© Springer-Verlag New York Inc. 1998

Authors and Affiliations

  • Verónica  Valverde-Garduño
    • 1
  • Patricio  Gariglio
    • 2
  • Lourdes  Gutiérrez
    • 1
  1. 1.Departamento de Virus y Cáncer, Centro de Investigaciones Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Avenida Universidad 655, Cuernavaca 62508, Morelos, MéxicoMX
  2. 2.Departamento de Genética y Biología Molecular, CINVESTAV—Instituto Politécnico Nacional, México, D.F. 07000, MéxicoMX

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