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

Abstract

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 (AEP) 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 Mn 2+ , a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas, in the presence of Mg 2+ , 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 Mn 2+ 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.

Identifying the role of PrimPol in TDF-induced toxicity and implications of its loss of function mutation in an HIV+ patient

“…Assuming that PrimPol may utilize either Mg 2+ or Mn 2+ in the cell 35,57 , the stabilization/destabilization effect may favor a particular mode for PrimPol. In agreement with this idea, a recent paper 58 demonstrated that in the presence of Mn 2+ , a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas, in the presence of Mg 2+ , the chemical step becomes rate limiting. The appearance of two melting minima for wild-type PrimPol, which correspond to the ZnF and the catalytic polymerase domain, may also aid in understanding the primase mechanism of PrimPol.…”

Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase

“…Thus, the active site of PrimPol, even after the binding of both ions, appears to be less tightly organized than Pol η’s and possibly more dependent on slow thermal motion to align the catalytic groups. The slow catalytic tuning might represent the ‘rate-limiting conformational transition step’ detected for PrimPol during a pre-steady-state kinetics study 48 .…”

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy

“…The active site of PrimPol has the following conserved key residues (compiled in Figure 7 A and Table 1 ) that have been studied in vitro : the three conserved carboxylates D 114 , E 116 , and D 280 , which are the residues that cause the covalent binding of the two-metal cations used to carry out the above mentioned nucleotidyl transferase reaction [ 12 , 77 , 107 ]. Mn 2+ is the preferred metal for PrimPol activities in vitro [ 12 , 75 , 77 , 108 , 109 , 110 ], despite having lesser fidelity than with Mg 2+ [ 75 , 109 , 110 ]. Mutations of the three carboxylates for alanine erase primase and polymerase activities [ 12 , 77 ].…”