Purification and properties of the 5'-3' exonuclease D10A mutant of DNA polymerase I from Streptococcus pneumoniae: a new tool for DNA sequencing.
A D10A mutation was introduced at the 5'-3' exonuclease domain of Streptococcus pneumoniae DNA polymerase I by site directed mutagenesis of the polA gene. Introduction of the mutation resulted in a drastic decrease of the 5'-3' exonucleolytic activity present in the wild-type enzyme. Moreover, the mutation at the D10 residue of the pneumococcal polymerase affected the dependency on metal activation of its 5'-3' exonucleolytic activity. These results provide experimental support for the proposed direct involvement of this Asp residue in a metal-assisted 5'-3' exonucleolytic reaction in type I-like bacterial DNA polymerases and related bacteriophage 5'-3' exonucleases. The D10A mutant polypeptide retained the polymerase activity of its parental enzyme, it is able to incorporate correctly nucleotides in a DNA template, and efficiently uses labeled and unlabeled nucleotides analogues in DNA sequencing by the dideoxy-chain-termination method. These characteristics convert this polymerase into a useful tool for manual and automatic sequencing.
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
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Amblar
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Sagner
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López
D004247:DNA / Q000378:metabolism; D004256:DNA Polymerase I / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D005092:Exonucleases / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013296:Streptococcus pneumoniae / Q000201:enzymology*
D011994:Recombinant Proteins; D004247:DNA; D004256:DNA Polymerase I; D005092:Exonucleases