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Open Access item La DNA polimerasa del bacteriófago phi 29: análisis mutacional de la interacción con la proteína terminal. Base estructural de la procesividad y la capacidad de desplazamiento de banda

Authors:Rodríguez García, Irene
Advisor:Vega, Miguel de
Salas, Margarita
Keywords:Bacteriófagos, Replicación de ADN
Issue Date:2006
Publisher:Universidad Autónoma de Madrid
Abstract:Bacteriophage φ29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication. φ29 DNA replication requires the formation of an heterodimer between the φ29 DNA polymerase and a free terminal protein (TP) molecule. During initiation, this polymerase catalyses the addition of dAMP to the OH group of Ser232 in the TP. It has been demonstrated that the same DNA polymerase that accomplishes initiation catalyses the elongation processively and coupled to strand displacement without the need of either processivity factors or helicase-like proteins. The φ29 DNA polymerase has served as model for the study of the biochemical features and of the structure-function relationships of DNA polymerases by means of an exhaustive mutational analysis of the enzyme, carried out over more than one decade. In this work, we have studied the functional role of several residues with a high degree of conservation among protein-primed DNA polymerases in specific reactions for these polymerases. Mutations introduced at residues Phe128, Glu161, Arg96, Lys110, Lys112, Arg113 and Lys114, belonging to the N-terminal domain of φ29 DNA polymerase, allow us to propose a contribution of those amino acids in establishing the appropriate interactions with DNA polymerase substrates, DNA and TP, to successfully accomplish the first steps of TPDNA replication. In addition, we analysed the role of two conserved residues, Lys305 and Tyr315, of φ29 DNA polymerase located at the insertion called TPR1 (Terminal Protein Region 1), only found in the subgroup of DNA polymerases that use a TP as primer. We found that these residues are involved in the correct positioning of the primer-terminus at the polymerase active site. Mutations at these residues produced a severe impairment in the ability to replicate φ29 TP-DNA. On the other hand, recent crystallographic studies of φ29 DNA polymerase have provided structural insights into its intrinsic processivity and ability to couple strand displacement to DNA synthesis. Biochemical analysis of a φ29 DNA polymerase deletion mutant lacking the TPR2 (Terminal Protein Region 2) subdomain demonstrates that it plays a critical role in processivity and strand displacement. Finally, we studied the possible molecular dynamics between the TPR2 and thumb subdomains of φ29 DNA polymerase during φ29 DNA replication
Description:Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, 24-11-2006
URI:http://hdl.handle.net/10261/8343
Appears in Collections:(CBM) Tesis

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