Thermodynamic Profiling Reveals DNA Polymerase Template Binding, Substrate Incorporation, and Exonuclease Function

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.