title: A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase.
abstract: Three conserved motifs (named A, B and C) have been proposed to form the polymerization active site in all classes of DNA-dependent polymerases. In eukaryotic-type (alpha-like) DNA polymerases, motif A is characterized by the consensus "Dx2SLYP". Mutants in phi29 DNA polymerase residue Tyr254 of this conserved motif had been previously shown to be affected in dNTP binding. Here, we show that a single substitution of Tyr254 into a valine residue enables the enzyme to incorporate ribonucleotide substrates, without affecting its wild-type affinity for dNTPs. Whereas the wild-type enzyme preferred dNTPs more than two million-fold over rNTPs, the mutation of Tyr254 into valine reduced the discrimination for rNTPs up to 1000-fold. In addition to this discrimination mechanism, based on sugar selection, phi29 DNA polymerase is very inefficient when extending an RNA primer terminus, allowing its exonucleolytic degradation. These results indicate that the Tyr254 of phi29 DNA polymerase is responsible for the discrimination against the 2'-OH group of an incoming ribonucleotide. This is the first time that the invariant tyrosine residue of motif A is involved in ribo- versus deoxyribonucleotide discrimination in an eukaryotic-type DNA polymerase.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, 28049, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Bonnin
authors.forename: J M
authors.initials: JM
authors.lastname: Lázaro
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; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012265:Ribonucleotides / Q000737:chemistry*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D012265:Ribonucleotides; D014443:Tyrosine; D004259:DNA-Directed DNA Polymerase