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

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.