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

Isothermal titration calorimetry (ITC) provides direct insight into the energetics of DNA polymerase function, including binding, catalysis, and exonuclease activity. We characterized a Phi29 mutant polymerase (SS_01) engineered to incorporate non-natural nucleotides in the presence of Mg2+, a function absent in the wild-type enzyme. ITC analyses revealed that SS_01 binding to the primed template was strongly influenced by metal ions. In the presence of Mg2+, the polymerase displayed tight binding (KD = 243 nM) and a clear exothermic signal, indicating activation of a large fraction of catalytically competent molecules. By contrast, in the presence of Ca2+, binding produced weaker exothermic signals (KD = 317 nM), suggesting less efficient binding complex formation. During dNTP- or oligonucleotide-tagged dNTP-driven polymerization, ITC profiles with Mg2+ exhibited pronounced endothermic heat changes, whereas with Ca2+, only minimal heat changes were observed. When binding only oligonucleotide-tagged dNTPs, the polymerases showed distinct thermodynamic behavior: in the presence of Mg2+, high substrate concentrations induced endothermic responses, while in the absence of catalytic ions, binding remained exothermic. Exonuclease activity monitored using unmodified oligonucleotides yielded strong exothermic signals in the presence of Mg2+ but weak responses in the presence of Ca2+, confirming strict ion dependence. Together, these data demonstrate that ITC directly captures the metal ion-dependent energetics of SS_01, providing mechanistic insight into its polymerization and exonuclease functions.