Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity

Autor: Sebastian Dorawa, Olesia Werbowy, Magdalena Plotka, Anna-Karina Kaczorowska, Joanna Makowska, Lukasz P. Kozlowski, Olafur H. Fridjonsson, Gudmundur O. Hreggvidsson, Arnthór Aevarsson, Tadeusz Kaczorowski
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 23, Iss 14, p 7945 (2022)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
DOI: 10.3390/ijms23147945
Popis: We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3′-5′ exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme’s activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme’s moderate thermal stability.
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