Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site
Recent crystallographic resolution of φ29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx2h motif of proofreading family B DNA polymerases. Single sub...
| 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:10486/709542 |
| Acceso en línea: | http://hdl.handle.net/10486/709542 https://dx.doi.org/10.1016/j.jmb.2009.06.068 |
| Access Level: | acceso abierto |
| Palabra clave: | ϕ29 DNA Polymerase 3′-5′ Exonuclease Site-Directed Mutagenesis ssDNA Binding Strand Displacement Biología y Biomedicina / Biología |
| Sumario: | Recent crystallographic resolution of φ29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx2h motif of proofreading family B DNA polymerases. Single substitution of φ29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx2hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at φ29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue |
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