title: Characterization and mapping of the pyrophosphorolytic activity of the phage phi 29 DNA polymerase. Involvement of amino acid motifs highly conserved in alpha-like DNA polymerases.
abstract: The phi 29 DNA polymerase, an alpha-like DNA polymerase, shows an inorganic pyrophosphate-dependent degradative activity with similar requirements to the corresponding one of Escherichia coli DNA polymerase I: (a) it requires a high concentration of inorganic pyrophosphate and is reversed by polymerization; (b) like DNA polymerization, it needs a duplex DNA with protruding 5' single-strand; (c) it acts in the 3' to 5' direction releasing free dNTPs, thus, it can be considered as the reversal of polymerization; (d) although a correctly base-paired 3' primer terminus is the preferred substrate, the pyrophosphorolytic activity is able to remove mismatched 3' ends. In agreement with the structural and functional model previously proposed for the phi 29 DNA polymerase, the analysis of point mutations has revealed that the pyrophosphorolytic activity, like the polymerization activity, is located at the C-terminal portion of the molecule, involving the amino acid motif YCDTD, highly conserved in alpha-like DNA polymerases. Furthermore, the analysis of phi 29 DNA polymerase mutants indicates that pyrophosphorolysis, like DNA polymerization, also requires an efficient translocation of the enzyme along the template.
authors.affiliation: Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
authors.forename: M A
authors.initials: MA
authors.lastname: Blasco
authors.forename: A
authors.initials: A
authors.lastname: Bernad
authors.forename: L
authors.initials: L
authors.lastname: Blanco
authors.forename: M
authors.initials: M
authors.lastname: Salas
mesh_terms: D001435:Bacteriophages / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D013379:Substrate Specificity; D013698:Templates, Genetic
chemical_list: D004279:DNA, Viral; D011756:Diphosphates; D004259:DNA-Directed DNA Polymerase