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.