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Nucleic Acids Res. 2002 Oct 1;30(19):4314-20.

Mutant Thermotoga neapolitana DNA polymerase I: altered catalytic properties for non-templated nucleotide addition and incorporation of correct nucleotides.

Author information

1
Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA and. Genecopoeia Inc., 15 Wormans Mill Court, Building D, Frederick, MD 21701, USA. swyang@genecopoeia.com

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.

PMID:
12364611
PMCID:
PMC140547
DOI:
10.1093/nar/gkf547
[Indexed for MEDLINE]
Free PMC Article

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