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.
Purification and properties of the 5′-3′ exonuclease D10A mutant of DNA polymerase I from Streptococcus pneumoniae: a new tool for DNA sequencing
스트렙토코쿠스 폐렴구균에서 DNA 중합효소 I의 5′-3′ 엑소뉴클레아제 D10A 돌연변이의 정제 및 특성: DNA 염기서열 분석을 위한 새로운 도구
[Category] 폐렴구균 감염증,
[Article Type] journal-article
[Source] pubmed
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