Differential Inhibition of Archaeal Pca DNA Polymerase by Monovalent Cations: Mechanistic Insights and Implications for PCR Fidelity.

This preprint presents a comparative investigation of ammonium (NH₄⁺) and potassium (K⁺) ion-mediated inhibition of recombinant Pyrobaculum calidifontis (Pca) DNA polymerase under PCR conditions. The enzyme was heterologously expressed in Escherichia coli, purified, and evaluated using PCR-based assays across a concentration range of 0–50 mM. Amplification products were analyzed by agarose gel electrophoresis and semi-quantitative densitometric analysis to estimate relative enzyme activity. Both ions exhibited concentration-dependent inhibition; however, distinct inhibitory profiles were observed. Ammonium ions induced a gradual decline in amplification efficiency, whereas potassium ions caused a markedly stronger inhibitory effect, with a sharper reduction in activity at intermediate and higher concentrations. A pronounced inhibitory threshold was observed between 20 and 30 mM, beyond which amplification efficiency decreased substantially. These findings demonstrate that monovalent cations exert differential effects on archaeal DNA polymerase activity and suggest that ion-specific modulation of enzyme-DNA interactions and Mg²⁺-dependent catalysis contributes to inhibition. This study provides a semi-quantitative framework for understanding ionic influences on polymerase function and offers practical insights for PCR buffer optimization and amplification fidelity.