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Functional analysis of HIV-1 reverse transcriptase motif C: site-directed mutagenesis and metal cation interaction
V Valverde-Garduño1, P Gariglio, L Gutiérrez
1Departamento de Virus y Cáncer, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, México. veronica@ibt.unam.mx
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.
Insights
Manganese enables HIV-1 reverse transcriptase (RT) to act as an RNA replicase. A mutation in motif C affects DNA polymerase activity and eliminates RNA replicase activity, suggesting a shared active site and common polymerase origin.
Area of Science:
- Enzymology
- Molecular Biology
- Virology
Background:
- Motif C is conserved in all polymerases and is implicated in catalysis and metal binding, suggesting a common evolutionary origin.
- Previous studies showed manganese alters nucleotide substrate specificity in some polymerases.
Purpose of the Study:
- To investigate if the active site responsible for incorporating nonspecific substrates is the same as that for specific substrates.
- To explore the role of manganese and motif C in HIV-1 reverse transcriptase (RT) activity.
Main Methods:
- Investigated the effect of manganese on HIV-1 RT's ability to incorporate ribonucleotides (rNTPs) using an RNA template.
- Introduced a D186H mutation in motif C of HIV-1 RT and assessed its impact on DNA polymerase and RNA replicase activities.
- Analyzed the metal ion preference of wild-type and mutant RT in the presence of magnesium and manganese.
Main Results:
- Manganese enabled HIV-1 RT to incorporate rNTPs with an RNA template, demonstrating RNA replicase activity.
- The D186H mutation in motif C significantly reduced DNA polymerase activity and abolished RNA replicase activity.
- Mutant RT showed altered metal ion preference, with reduced activity in magnesium but increased relative activity in manganese compared to wild-type.
Conclusions:
- Both DNA polymerase and RNA replicase activities of HIV-1 RT depend on the same active site, involving residue D186 in motif C.
- Residue D186 is crucial for metal ion binding and is essential for both specific DNA synthesis and manganese-dependent RNA replication.
- These findings support the hypothesis that all polymerases evolved from a common ancestor containing at least motif C.