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 Biotechnoljournal_title
Journal of biotechnologyauthors
Amblar M,Sagner G,López Pdoi
10.1016/s0168-1656(98)00071-6subject
Has Abstractpub_date
1998-07-30 00:00:00pages
17-27issue
1eissn
0168-1656issn
1873-4863pii
S0168-1656(98)00071-6journal_volume
63pub_type
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