title: Purification and properties of the 5'-3' exonuclease D190-->a mutant of DNA polymerase I from Streptococcus pneumoniae.
abstract: A D190-->A mutation was introduced at the 5'-3' exonuclease domain of Streptococcus pneumoniae DNA polymerase I by site-directed mutagenesis of the polA gene. Comparison of the S. pneumoniae DNA polymerase I, its polymerase domain, and the [Ala190]exonuclease mutant revealed that the mutant polypeptide retains the polymerase activity of the parental enzyme and displayed the strand-displacement activity of its polymerase domain. However, introduction of the mutation resulted in a 2500-fold reduction of the 5'-3' exonuclease catalytic rate compared with the wild-type enzyme. Moreover, the mutation at the Asp190 residue of the pneumococcal polymerase affected the dependency on metal activation of its 5'-3' exonucleolytic activity. These results provide experimental support for a direct involvement of this aspartic acid residue in a metal-assisted 5'-3' exonucleolytic reaction in type-I-like bacterial DNA polymerases and related bacteriophage 5'-3' exonucleases.
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Amblar
authors.forename: P
authors.initials: P
authors.lastname: López
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004256:DNA Polymerase I / Q000235:genetics*; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology*
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008345:Manganese; D004247:DNA; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V