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

Abstract

Tt72 DNA polymerase is a newly characterized PolA-type thermostable enzyme derived from the Thermus thermophilus 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 lacZα 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.

Keywords: 3′→5′ exonuclease activity; PolA-type enzyme; molecular cloning; proofreading.

Figure 1

Characterization of 3′→5′ exonuclease activity of the Tt72 DNA polymerase. The enzyme’s activity was assessed by measuring the radioactivity released from labeled λ/HindIII DNA fragments. (a) 3′→5′ exo activity of Tt72 DNA polymerase after 10 min heat treatment at temperatures ranging from 55 °C to 95 °C. (b) Effect of dNTP concentration on the 3′→5′ exo activity of the Tt72 DNA polymerase. Error bars indicate the mean  ±  standard deviations. Each experiment was conducted in triplicate.

Figure 2

Schematic illustration of DNA 3′/5′-ends polishing with Tt72 DNA polymerase. Cutting sites are indicated by triangles.

Figure 3

Schematic illustration of PCR product 3-overhang polishing with Tt72 DNA polymerase.