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Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/37679
Title: φ29 DNA Polymerase–Terminal Protein Interaction. Involvement of Residues Specifically Conserved Among Protein-primed DNA Polymerases
Authors: Rodríguez García, Irene; Lázaro, José M.; Salas, Margarita; Vega, Miguel de
Keywords: DNA polymerase
Site-directed mutagenesis
Linear DNA replication
Terminal protein
Issue Date: 2-Mar-2004
Publisher: Elsevier
Citation: Journal of Molecular Biology 337(4): 829-841 (2004)
Abstract: By multiple sequence alignments of DNA polymerases from the eukaryotic-type (family B) subgroup of protein-primed DNA polymerases we have identified five positively charged amino acids, specifically conserved, located N-terminally to the (S/T)Lx2h motif. Here, we have studied, by site-directed mutagenesis, the functional role of φ29 DNA polymerase residues Arg96, Lys110, Lys112, Arg113 and Lys114 in specific reactions dependent on a protein-priming event. Mutations introduced at residues Arg96, Arg113 and Lys114 and to a lower extent Lys110 and Lys112, showed a defective protein-primed initiation step. Analysis of the interaction with double-stranded DNA and terminal protein (TP) displayed by mutant derivatives R96A, K110A, K112A, R113A and K114A allows us to conclude that φ29 DNA polymerase residue Arg96 is an important DNA/TP-ligand residue, essential to form stable DNA polymerase/DNA(TP) complexes, while residues Lys110, Lys112 and Arg113 could be playing a role in establishing contacts with the TP-DNA template during the first step of DNA replication. The importance of residue Lys114 to make a functionally active DNA polymerase/TP complex is also discussed. These results, together with the high degree of conservation of those residues among protein-primed DNA polymerases, strongly suggest a functional role of those amino acids in establishing the appropriate interactions with DNA polymerase substrates, DNA and TP, to successfully accomplish the first steps of TP-DNA replication.
Publisher version (URL): http://dx.doi.org/10.1016/j.jmb.2004.02.018
URI: http://hdl.handle.net/10261/37679
ISSN: 0022-2836
DOI: 10.1016/j.jmb.2004.02.018
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