Divalent Cations Alter the Rate-Limiting Step of PrimPol-Catalyzed DNA Elongation

PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using Enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn²⁺, a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas in the presence of Mg²⁺, the chemical step becomes rate limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn²⁺ concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase.

DNA polymerase; DNA replication; enzyme kinetics; enzyme mechanism; enzymology.