Precise and Accurate DNA-3′/5-Ends Polishing with Thermus thermophilus Phage vb_Tt72 DNA Polymerase

Tt72 DNA polymerase is a newly characterized PolA-type thermostable enzyme derived from the <i>Thermus thermophilus</i> phage vB_Tt72. The enzyme demonstrates strong 3'→5' exonucleolytic proofreading activity, even in the presence of 1 mM dNTPs. In this study, we examined how the exonucleolytic activity of Tt72 DNA polymerase affects the fidelity of DNA synthesis. Using a plasmid-based <i>lacZα</i> gene complementation assay, we determined that the enzyme's mutation frequency was 2.06 × 10^-3, corresponding to an error rate of 1.41 × 10^-5. For the exonuclease-deficient variant, the mutation frequency increased to 6.23 × 10^-3, with an associated error rate of 4.29 × 10^-5. The enzyme retained 3'→5' exonucleolytic activity at temperatures up to 70 °C but lost it after 10 min of incubation at temperatures above 75 °C. Additionally, we demonstrated that Tt72 DNA polymerase efficiently processes 3'/5'-overhangs and removes a single-nucleotide 3'-dA overhang from PCR products at 55 °C. These characteristics make Tt72 DNA polymerase well suited for specialized molecular cloning applications.