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

By Shu-Wei Yang, Mekbib Astatke, Jason Potter and Deb K. Chatterjee


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

Topics: Articles
Publisher: Oxford University Press
Year: 2002
DOI identifier: 10.1093/nar/gkf547
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Provided by: PubMed Central
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