Family A DNA polymerase (K4Poll) from Thermotoga petrophila K4 was obtained as a recombinant form, and the enzyme characteristics were analyzed. K4Poll showed thermostable DNA-dependent DNA polymerase activity with 3'-5' exonuclease activity but no detectable RNA-dependent DNA polymerase activity. Its tertiary structure was speculated by in silico modeling to understand the binding situation between K4Poll and template DNA. Nine amino acids in the 3'-5' exonuclease domain are predicted to be involved in DNA/RNA distinction by steric interference with the 2' hydroxy group of ribose. To allow K4Poll to accept RNA as the template, mutants were constructed focusing on the amino acids located around the 2' hydroxyl group of the bound ribose. The mutants in which Thr326, Leu329, Gln384, Phe388, Met408, or Tyr438 was replaced with Ala (designated as T326A, L329A, Q384A, F388A, M408A, or Y438A, respectively) showed RNA-dependent DNA polymerase activity. All the mutants showed reduced 3'-5' exonuclease activity, suggesting that gain of reverse transcriptase activity is correlated with loss of 3'-5' exonuclease activity. In particular, the mutants enabled direct DNA amplification in a single tube format from structured RNA that was not efficiently amplified by retroviral reverse transcriptase. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.