Insights into the Molecular Mechanism of Polymerization and Nucleoside Reverse Transcriptase Inhibitor Incorporation by Human PrimPol

Abstract

Human PrimPol is a newly identified DNA and RNA primase-polymerase of the archaeo-eukaryotic primase (AEP) superfamily and only the second known polymerase in the mitochondria. Mechanistic studies have shown that interactions of the primary mitochondrial DNA polymerase γ (mtDNA Pol γ) with nucleoside reverse transcriptase inhibitors (NRTIs), key components in treating HIV infection, are a major source of NRTI-associated toxicity. Understanding the interactions of host polymerases with antiviral and anticancer nucleoside analog therapies is critical for preventing life-threatening adverse events, particularly in AIDS patients who undergo lifelong treatment. Since PrimPol has only recently been discovered, the molecular mechanism of polymerization and incorporation of natural nucleotide and NRTI substrates, crucial for assessing the potential for PrimPol-mediated NRTI-associated toxicity, has not been explored. We report for the first time a transient-kinetic analysis of polymerization for each nucleotide and NRTI substrate as catalyzed by PrimPol. These studies reveal that nucleotide selectivity limits chemical catalysis while the release of the elongated DNA product is the overall rate-limiting step. Remarkably, PrimPol incorporates four of the eight FDA-approved antiviral NRTIs with a kinetic profile distinct from that of mtDNA Pol γ that may manifest in toxicity.

FIG 1

The binding affinity of PrimPol for the DNA primer-template substrates as determined by EMSA. Affinities of PrimPol for the DNA primer-template substrates (where D20, D22, D23 and D30 are primers and D45 is the template) (see Table S1 in the supplemental material for sequences) used in the study were determined by gel mobility shift assay. DNA primer-template (3 nM) was mixed with 0 to 5 μM PrimPol protein and separated by electrophoresis. Quantitative analysis of autoradiograms was used to calculate the fraction of DNA primer-template bound to determine the K_d.

FIG 2

Transient-state kinetic analysis of polymerization by PrimPol. Single-nucleotide incorporation of dCTP into the DNA primer-template D23-D45 under pre-steady-state burst conditions as catalyzed by PrimPol demonstrates biphasic kinetics: a burst of product formation at a rate of 0.36 ± 0.04 s⁻¹ followed by a slower, linear phase with a rate of 0.046 ± 0.006 s⁻¹.

FIG 3

Transient-state kinetic analysis for correct nucleotide incorporation as catalyzed by PrimPol. The concentration dependence of correct nucleotide incorporation on rate was fit to a quadratic equation to generate rate constants k_pol and K_d for dATP, dTTP, dCTP, and dGTP, as indicated.

FIG 4

PrimPol readily misincorporates dATP at n + 2 (22 nt) opposite a template dG below and above the K_d for dATP. Correct dATP incorporation is observed at n + 1 (21 nt) opposite a template dT while dATP misincorporation is observed at n + 2 (22 nt) opposite a template dG. PrimPol misincorporates at 1 μM (15 and 60 s), 5 μM (3, 10, and 30 s), and 25 μM dATP (5 s), even before all of substrate n (20 nt) is turned over.

FIG 5

PrimPol incorporates four of the eight FDA-approved NRTIs. Depicted here are the structures of the metabolically active triphosphate forms of the four NRTIs: carbovir triphosphate (CBV-TP) which is the active metabolite of abacavir, didanosine triphosphate, which is the active metabolite of didanosine, zalcitabine TP, and zidovudine TP.

FIG 6

Transient-state kinetic analysis for NRTI incorporation as catalyzed by PrimPol. The concentration dependence (amplitude for AZT) of NRTI incorporation on rate was fit to a quadratic equation to generate rate constants k_pol and K_d for ddATP, AZT-TP, ddCTP, and CBV-TP, as indicated.