Cloning and Functional Characterization of AhyAP-T65Lig, an ATP-Dependent DNA Ligase from Trabzonvirus AP-T65.

Abstract

DNA ligases are essential enzymes in all living organisms and viruses. They play critical roles in genome maintenance and recombination by sealing single-strand nicks and repairing damaged DNA. Due to these features, DNA ligases are widely used in gene cloning applications and in vitro processing of DNA. In this study, the DNA ligase-encoding gene from Trabzonvirus AP-T65 was cloned and sequenced. AP-T65 DNA ligase (AhyAP-T65Lig) shared 51.81% sequence similarity with the DNA ligase of Aeromonas phage BUCT695, and 53.35% with that of Salmonella phage PKM.Hi.22.6. AhyAP-T65Lig was cloned into pET-28a(+) expression vector, overexpressed and purified. The ligation efficiency of AhyAP-T65Lig (41.1 kDa) was evaluated using ligation-mediated PCR and RT-PCR. AhyAP-T65Lig was active between 4 and 37 °C, with an optimum temperature of 4 °C and an optimum pH of 8.0. Maximum activity was observed in the presence of 1 mM ATP, 10 mM DTT, 10 mM MgCl_2, and 1 mM NaCl. Sticky-ended, linearized pGEM-T Easy and pET-28a(+) vectors were circularized by AhyAP-T65Lig. The enzyme retained its activity for an extended period at both 4 and - 20 °C. The low-temperature optimum, high in vitro yield, and long shelf-life of AhyAP-T65Lig make it a promising enzyme for biotechnological applications.