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

Nucleic Acids Res. 2002 Oct 1;30(19):4314-20. doi: 10.1093/nar/gkf547.


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.

MeSH terms

  • Amino Acid Sequence
  • Catalysis
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism*
  • Gram-Negative Anaerobic Straight, Curved, and Helical Rods / enzymology*
  • Mutation
  • Mutation, Missense
  • Sequence Homology, Amino Acid
  • Substrate Specificity


  • DNA Polymerase I