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¹Research Center of Molecular Diagnostics and Sequencing, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518000, China.

International Journal of Molecular Sciences

|December 30, 2025

Summary

Isothermal titration calorimetry reveals metal ion-dependent energetics of a novel DNA polymerase (SS_01). This mutant incorporates non-natural nucleotides, with Mg2+ enabling tighter binding and efficient catalysis compared to Ca2+.

Keywords:

Phi29 DNA polymerases exonuclease activity incorporation isothermal titration calorimetry template binding thermodynamic profiling

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Area of Science:

Biochemistry
Molecular Biology
Enzyme Kinetics

Background:

Isothermal titration calorimetry (ITC) is a powerful biophysical technique for studying enzyme mechanisms.
DNA polymerases are crucial enzymes involved in DNA replication and repair.
Metal ions play critical roles in the catalytic activity of DNA polymerases.

Purpose of the Study:

To characterize the metal ion-dependent energetics of a Phi29 mutant DNA polymerase (SS_01) engineered for non-natural nucleotide incorporation.
To elucidate the mechanistic insights into the polymerization and exonuclease functions of SS_01 using ITC.
To compare the binding and catalytic efficiencies of SS_01 in the presence of Mg2+ versus Ca2+.

Main Methods:

Isothermal titration calorimetry (ITC) was employed to measure the binding and catalytic energetics.
The study utilized a Phi29 mutant polymerase (SS_01) capable of incorporating non-natural nucleotides.
Experiments were conducted in the presence of different metal ions (Mg2+ and Ca2+) and various substrates (dNTPs, oligonucleotide-tagged dNTPs, unmodified oligonucleotides).

Main Results:

SS_01 exhibited tight binding (KD = 243 nM) with Mg2+, showing a clear exothermic signal indicative of catalytically competent molecules.
Binding with Ca2+ resulted in weaker exothermic signals (KD = 317 nM), suggesting less efficient complex formation.
Polymerization assays with Mg2+ showed pronounced endothermic heat changes, while Ca2+ yielded minimal changes.
Exonuclease activity was strongly exothermic with Mg2+ but weak with Ca2+, highlighting strict ion dependence.

Conclusions:

ITC directly captures the metal ion-dependent energetics of SS_01 DNA polymerase.
The findings provide mechanistic insights into the polymerization and exonuclease functions of SS_01.
Metal ion choice (Mg2+ vs. Ca2+) significantly impacts the binding affinity, catalytic efficiency, and overall thermodynamic behavior of the enzyme.