Purification and properties of the 5'-3' exonuclease D10A mutant of DNA polymerase I from Streptococcus pneumoniae: a new tool for DNA sequencing.

Abstract:

: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.

journal_name

J Biotechnol

journal_title

Journal of biotechnology

authors

Amblar M,Sagner G,López P

doi

10.1016/s0168-1656(98)00071-6

subject

Has Abstract

pub_date

1998-07-30 00:00:00

pages

17-27

issue

1

eissn

0168-1656

issn

1873-4863

pii

S0168-1656(98)00071-6

journal_volume

63

pub_type

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