Functional analysis of HIV-1 reverse transcriptase motif C: site-directed mutagenesis and metal cation interaction.
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.
Valverde-Garduño
V
V
Departamento de Virus y Cáncer, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, México. veronica@ibt.unam.mx
Gariglio
P
P
Gutiérrez
L
L
D001665:Binding Sites; D002412:Cations / Q000378:metabolism; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000187:drug effects / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D008345:Manganese / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects
D002412:Cations; D018894:Reverse Transcriptase Inhibitors; D008345:Manganese; D012324:RNA-Dependent RNA Polymerase; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase