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Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/38987
Title: φ29 DNA Polymerase Residue Phe128 of the Highly Conserved (S/T)Lx2h Motif is Required for a Stable and Functional Interaction with the Terminal Protein
Authors: Rodríguez García, Irene; Lázaro, José M.; Salas, Margarita; Vega, Miguel de
Keywords: DNA polymerase
Site directed mutagenesis
Issue Date: 3-Jan-2003
Publisher: Elsevier
Citation: Journal of Molecular Biology 325(1): 85-97 (2003)
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. By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx2h motif, and a glutamate belonging to the Exo III motif. Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of φ29 DNA polymerase. Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As a consequence, very few initiation products were obtained, and essentially no transition products were detected. Interestingly, φ29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules. These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps.
Publisher version (URL): http://dx.doi.org/10.1016/S0022-2836(02)01130-0
URI: http://hdl.handle.net/10261/38987
ISSN: 0022-2836
DOI: 10.1016/S0022-2836(02)01130-0
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