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.
D000595:Amino Acid Sequence; D002384:Catalysis; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology*; D009154:Mutation; D020125:Mutation, Missense; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004256:DNA Polymerase I