Characterization of DNA Polymerase from <i>Thermus thermophilus</i> MAT72 Phage Tt72

• Main Authors: Sebastian Dorawa, Magdalena Plotka, Anna-Karina Kaczorowska, Olafur H. Fridjonsson, Gudmundur O. Hreggvidsson, Arnthor Aevarsson, Tadeusz Kaczorowski
• Format: Article
• Language: English
• Published: MDPI AG 2020-06-01
• Series: Proceedings
• Subjects: Thermus phage; DNA polymerase; 3′ → 5′ exonuclease
• Online Access: https://www.mdpi.com/2504-3900/50/1/38

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 <i>Thermus thermophilus</i><i> </i>MAT72. An <i>in </i><i>silico</i><i> </i>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 <i>T. thermophilus</i><i> </i>phages: ΦYS40 (91%) and ΦTMA (90%). The gene coding for Tt72 DNA polymerase was cloned and overexpressed in <i>E. coli</i>. The Tt72 <i>polA</i><i> </i>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 <i>T. thermophilus</i><i> </i>genomic DNA (69.49%). The Tt72 <i>polA</i><i> </i>gene codes for a 703-aa protein with a predicted molecular weight of 80,477. The enzyme was overproduced in <i>E. coli</i>, 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 Mg²⁺. Furthermore, the Tt72 DNA polymerase shows strong 3′ → 5′ exonucleolytic activity.