Magnesium Concentration Modulates Replication Slippage of Mesophilic and Thermophilic DNA Polymerases In Vitro

Replication slippage at repetitive DNA sequences generates insertions and deletions that drive genomic instability. Although magnesium ions are essential cofactors for DNA polymerase activity, their role in modulating slippage fidelity remains unclear. Using an in vitro primer extension assay on a single-stranded template carrying direct repeats flanking a hairpin-forming inverted repeat, we investigated the effect of Mg²⁺ concentration on slippage produced by mesophilic (T4 Pol, T7 Pol, E. coli pol I Klenow fragment, pol I KF exo^-, and pol III holoenzyme) and thermophilic (Taq pol and Pfu pol) DNA polymerases. We show that Mg²⁺ modulates slippage frequency in a polymerase-dependent manner, as follows: low concentrations suppress slippage in T7 Pol, pol I KF, pol III HE, and Taq Pol, whereas T4 Pol and Pfu Pol slip at all productive concentrations. Mechanistically, Mg²⁺ modulates strand displacement activity, and polymerases that acquire enhanced strand displacement at intermediate concentrations show a corresponding reduction in slippage. Proofreading activity had no detectable effect on slippage frequency. These findings reinforce the inverse correlation between strand displacement activity and slippage propensity and suggest that physiological free Mg²⁺ levels may help suppress slippage in vivo.