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

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 (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: phi YS40 (91%) and phi TMA (90%). The Tt72 polA gene does not complement the Escherichia coli polA(-) 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 degrees C. Above 60 degrees C, the rapid loss of function follows with no activity > 75 degrees C. However, during heat treatment (10 min at 75 degrees C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at T-m = 74.6 degrees C (Delta H-cal = 2.05 x 10(4) cal mol(-1)) and circular dichroism spectra > 60 degrees C indicate the enzyme's moderate thermal stability.

Automated summary

We performed a structural and functional analysis of the DNA polymerase from the thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme showed low sequence identity to DNA polymerases from other families, but high similarity to two uncharacterized DNA polymerases from other T. thermophilus phages. We found that the enzyme exists as a monomer in solution and has optimal activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis revealed critical residues for the enzyme's activity. Despite its source, the enzyme itself did not exhibit high thermoresistance, but could be protected against thermal inactivation by specific compounds. Our study also demonstrated that the enzyme has moderate thermal stability.