ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation
DNA polymerases require two acidic residues to coordinate metal ions A and B at their polymerisation active site during catalysis of nucleotide incorporation. Crystallographic resolution of φ{symbol}29 DNA polymerase ternary complex showed that metal B coordination also depends on the carbonyl group...
| Autores: | , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 2009 |
| País: | España |
| Institución: | Universidad Autónoma de Madrid |
| Repositorio: | Biblos-e Archivo. Repositorio Institucional de la UAM |
| Idioma: | inglés |
| OAI Identifier: | oai:repositorio.uam.es:10486/709525 |
| Acceso en línea: | http://hdl.handle.net/10486/709525 https://dx.doi.org/10.1016/j.jmb.2009.10.061 |
| Access Level: | acceso abierto |
| Palabra clave: | Metal Ligand Polymerisation Active Site Protein-Priming Strand Displacement ϕ29 DNA Polymerase Biología y Biomedicina / Biología |
| Sumario: | DNA polymerases require two acidic residues to coordinate metal ions A and B at their polymerisation active site during catalysis of nucleotide incorporation. Crystallographic resolution of φ{symbol}29 DNA polymerase ternary complex showed that metal B coordination also depends on the carbonyl group of Val250 that belongs to the highly conserved Dx2SLYP motif of eukaryotic-type (family B) DNA polymerases. In addition, multiple sequence alignments have shown the specific conservation of this residue among the DNA polymerases that use a protein as primer. Thus, to ascertain its role in polymerisation, we have analysed the behaviour of single mutations introduced at the corresponding Val250 of φ{symbol}29 DNA polymerase. The differences in nucleotide binding affinity shown by mutants V250A and V250F with respect to the wild-type DNA polymerase agree to a role for Val250 as a metal B-dNTP complex ligand. In addition, mutant V250F was severely affected in φ{symbol}29 DNA replication because of a large reduction in the catalytic efficiency of the protein-primed reactions. In the light of the φ{symbol}29 DNA polymerase structures, a role for Val250 residue in the maintenance of the proper architecture of the enzyme to perform the protein-primed reactions is also proposed |
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