Characterization of DNA Polymerase from Thermus thermophilus MAT72 Phage Tt72

• Published in: Proceedings
• Main Authors: Sebastian Dorawa, Magdalena Plotka, Anna-Karina Kaczorowska, Olafur H. Fridjonsson, Gudmundur O. Hreggvidsson, Arnthor Aevarsson, Tadeusz Kaczorowski
• Format: Article in Journal/Newspaper
• Language: English
• Published: MDPI AG 2020
• Subjects: Thermus phage; DNA polymerase; 3′ → 5′ exonuclease; General Works; A; Talon; Iceland
• Online Access: https://doi.org/10.3390/proceedings2020050038; https://doaj.org/article/23f4499e29b8422fa99372253cd1c2f8

Thermophilic phages are recognized as an untapped source of thermostable enzymes relevant in biotechnology; however, their biology is poorly explored. This has led us to start a project aimed at investigating thermophilic phages isolated from geothermal areas of Iceland. In this study, we present a structural and functional analysis of the DNA polymerase of phage Tt72, which infects thermophilic bacterium Thermus thermophilus MAT72. An in silico analysis of the Tt72 phage genome revealed the presence of a 2112-bp open reading frame (ORF) encoding protein homologous to the members of the A family of DNA polymerases. It contains a conserved nucleotidyltransferase domain and a 3′ → 5′ exonuclease domain but lacks the 5′ → 3′ exonuclease domain. The amino acid sequence of Tt72 DNA polymerase shows high similarity to two as yet uncharacterized DNA polymerases of T. thermophilus phages: ΦYS40 (91%) and ΦTMA (90%). The gene coding for Tt72 DNA polymerase was cloned and overexpressed in E. coli . The Tt72 polA gene is composed of 2112 nucleotides. The overall G+C content of this gene is 31.58%, which is lower than the G+C content of T. thermophilus genomic DNA (69.49%). The Tt72 polA gene codes for a 703-aa protein with a predicted molecular weight of 80,477. The enzyme was overproduced in E. coli , purified by heat treatment, followed by HiTrap TALON column and HiTrap Heparin HP column chromatography, then biochemically characterized. The optimum activity was found at 55 °C, pH 8.5, 25 mM KCl, and 0.5 mM Mg 2+ . Furthermore, the Tt72 DNA polymerase shows strong 3′ → 5′ exonucleolytic activity.