[Characterization of a DNA polymerase fused with a DNA binding domain of colicin].

DNA polymerase, which was discovered from a thermophilic aquatic bacterium (), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. DNA polymerase lacking the 5'→3' exonuclease domain (Δ) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of Δ. To this end, we fused dCE with Δ and observed a significant improvement in the processivity of the resulting dCE-Δ compared to DNA polymerase and dCE-. Furthermore, its reverse transcriptase activity was also higher than that of Δ. The most notable improvement was observed in dCE8-Δ, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of DNA polymerase by fusing Δ DNA polymerase with dCE. This approach provides a novel approach for modifying DNA polymerase and holds potential for the development of improved variants of DNA polymerase.