Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol

Publication date: Available online 25 November 2016
Source:DNA Repair
Author(s): E. John Tokarsky, Petra C. Wallenmeyer, Kenneth K. Phi, Zucai Suo
Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn²⁺-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn²⁺, rather than Mg²⁺, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn²⁺ and Mg²⁺, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn²⁺ than with Mg²⁺. Notably, the substitution fidelity of PrimPol in the presence of Mn²⁺ was determined to be in the range of 3.4×10⁻² to 3.8×10⁻¹, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1,800 with Mn²⁺ and 150-4,500 with Mg²⁺, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).

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