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

ABSTRACT: 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 f

SUBMITTER: Sun Y 

PROVIDER: S-EPMC12732654 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature