Nucleic Acids Research, 2002, Vol. 30, No. 19 4314-4320
© 2002 Oxford University Press
Mutant Thermotoga neapolitana DNA polymerase I: altered catalytic properties for non-templated nucleotide addition and incorporation of correct nucleotides
Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA and 1 Genecopoeia Inc., 15 Wormans Mill Court, Building D, Frederick, MD 21701, USA
*To whom correspondence should be addressed at present address: SAIC-Frederick, National Cancer Institute at Frederick, Protein Expression Laboratory, Frederick, MD 21702, USA. Tel: +1 301 846 6893; Fax: +1 301 846 7390; Email: chatterjee{at}ncifcrf.gov
Correspondence may also be addressed to Shu-Wei Yang. Email: swyang{at}genecopoeia.com
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.