Mutant Thermotoga neapolitana DNA polymerase I: altered catalytic properties for non-templated nucleotide addition and incorporation of correct nucleotides.
Nucleic Acids Res
; 30(19): 4314-20, 2002 Oct 01.
Article
em En
| MEDLINE
| ID: mdl-12364611
ABSTRACT
Thermotoga neapolitana (Tne) DNA polymerase belongs to the DNA polymerase I (Pol I) family. The O-helix region of these polymerases is involved in dNTP binding and also plays a role in binding primer-template during DNA synthesis. Here we report that mutations in the O-helix region of Tne DNA polymerase (Arg722 to His, Tyr or Lys) almost completely abolished the enzyme's ability to catalyze the template-independent addition of a single base at the 3'-end of newly synthesized DNA in vitro. The mutations did not significantly affect the DNA polymerase catalytic activity and reduced base misinsertions 5- to 50-fold. The same Arg722 mutations dramatically increased the ability of the enzyme's 3'-->5' exonuclease to remove mispaired 3' bases in a primer extension assay. These mutant DNA polymerases can be used to accurately amplify target DNA in vitro for gene cloning and genotyping analysis.
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Base de dados:
MEDLINE
Assunto principal:
Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos
/
DNA Polimerase I
Idioma:
En
Ano de publicação:
2002
Tipo de documento:
Article