title: Role of the LEXE motif of protein-primed DNA polymerases in the interaction with the incoming nucleotide.
abstract: The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding ϕ29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg(2+) ions, a reaction highly improved when Mn(2+) was used as metal activator. These results, together with previous crystallographic resolution of ϕ29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg(2+). Recovery of both reactions with Mn(2+) supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP.
authors: From the Instituto de Biología Molecular "Eladio Viñuela" (Consejo Superior de Investigaciones Científicas), Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), C/Nicolás Cabrera 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
authors: Eugenia
authors: E
authors: Santos
authors: José M
authors: JM
authors: Lázaro
authors: Patricia
authors: P
authors: Pérez-Arnaiz
authors: Margarita
authors: M
authors: Salas
authors: Miguel
authors: M
authors: de Vega
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D017103:Bacillus Phages / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain / Q000502:physiology; D018360:Crystallography, X-Ray; D004261:DNA Replication / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D014764:Viral Proteins; D008345:Manganese; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase; D008274:Magnesium
keywords: Bacteriophage; DNA Polymerase; DNA Replication; Nucleic Acid Synthesis; Viral Polymerase