We studied the 5' ↑ 3' exonuclease activity of Bacillus caldotenax DNA polymerase by site-directed mutagenesis. Among seven mutants constructed, two mutant DNA polymerases with an amino acid substitution of Glyl84 ↑ Asp or Glyl92 ↑ Asp were confirmed to be deficient in this exonuclease. The two positions corresponded to those of the Escherichia coli DNA polymerase I mutants defective in 5' ↑ 3' exonuclease, polA480ex and polA214. These results provide experimental support for the proposed amino acid sequence essential for the 5' ↑ 3' exonuclease activity associated with eubacterial polymerase I-like DNA polymerases (family A), including E.coli and Thermits aquaticus.
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