Molecular dissection of the domain architecture and catalytic activities of human PrimPol

Keen, Benjamin A.; Jóźwiakowski, Stanisław K.; Bailey, Laura J.; Bianchi, Julie; Doherty, Aidan J.

doi:10.1093/nar/gku214

Cited by 96 publications

(215 citation statements)

References 42 publications

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“…Recent studies indicate that PrimPol has a weaker affinity for DNA primer-templates than other polymerases and a preference for the cofactor manganese (20,21,24). It was important to provide a quantitative assessment of equilibrium dissociation constants for DNA binding as an essential component of defining appropriate burst and single-turnover conditions for incorporation studies.…”

Section: Resultsmentioning

confidence: 99%

“…The equilibrium dissociation constant (K d ) of all DNA primer-template substrates for PrimPol were determined using electrophoretic mobility shift assays (EMSAs) as previously described (21,24) but with minor modifications. Various active-site concentrations of PrimPol (0 to 5 M) in 10 mM bis-Tris propane-HCl (pH 7.0), 1 mM DTT, and 10 mM MnCl 2 or MgCl 2 were incubated with 3 nM DNA primer-template substrate in a final volume of 20 l for 60 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…AZT-TP is a component of the fixed-dose combinations lamivudine-zidovudine (Combivir) and abacavir sulfate-lamivudine-zidovudine (Trizivir), and CBV-TP is also a component of Trizivir, abacavir-lamivudine (Epzicom), and a recently FDA-approved combination therapy with dolutegravir and lamivudine (Triumeq). Although it may be assumed that PrimPol performs fewer incorporation events than the primary replicative nuclear polymerases ␣, ε, and ␦ and mtDNA Pol ␥, NRTI incorporation by PrimPol at lesions, including 8oxoG (15,20,21), abasic sites (15), (6-4)pp (16,21), and CPDs (16), can result in DNA breaks, genomic instability, and ultimately apoptosis (39). Our findings highlight one possible alternative mechanism of antiviral toxicity for consideration in the clinical use of these drugs and future nucleoside analog development.…”

Section: Tablementioning

confidence: 99%

“…Importantly and in contrast to mtDNA Pol ␥, PrimPol also lacks a proofreading mechanism for excision of incorporated NRTIs. Understanding the consequences of PrimPol's inability to remove incorporated NRTIs in the context of its role in restarting replication forks and bypassing lesions, particularly in the mitochondrial genome, is important for future investigations (15,16,20,21 (14,42) or the C-terminal zinc finger domain, implicated in PrimPol's fidelity (21), play in regulating polymerization and nucleotide selectivity. The mtDNA Pol ␥ holoenzyme contains a catalytic subunit, Pol ␥A, and a dimeric accessory subunit, Pol ␥B, that enhance activity of the catalytic subunit (27,43).…”

Section: Tablementioning

confidence: 99%

“…Notably, it lacks 3=-to-5= exonuclease activity and preferentially binds the cofactor manganese (15,20). PrimPol functions in replication restart downstream of stalled replication forks (16) and can perform translesion synthesis, bypassing oxidative and UV-induced lesions, including 8-oxoguanine (8oxoG) (15,20,21), abasic sites (15), (6-4) pyrimidinepyrimidone photoproducts [(6-4)pp] (16, 21), and cyclobutane pyrimidine dimers (CPDs) (16). PrimPol helps to maintain mtDNA copy numbers and replication rates and can utilize both deoxynucleoside triphosphates (dNTPs) and NTPs (15).…”

mentioning

confidence: 99%

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

Mislak

Anderson

2016

Antimicrob Agents Chemother

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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 ␥ (mtDNA Pol ␥) 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 PrimPolmediated 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 ␥ that may manifest in toxicity.N ucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antivirals that target the human immunodeficiency virus (HIV) polymerase, reverse transcriptase (RT). All FDA-approved NRTIs are nucleoside analogs that lack a 3=-hydroxyl moiety to terminate DNA chain extension upon incorporation by RT into the growing proviral DNA. While significant health advances have been achieved with the use of NRTIs, the necessity for lifelong treatment to control HIV infection is limited by NRTI-associated toxicities that arise from virus-versus-host polymerase selectivity wherein NRTIs also serve as substrates for host polymerases (1, 2).The most severe NRTI-associated toxicities predominantly manifest in mitochondrial dysfunction (1-6) and are attributed primarily to incorporation by the human mitochondrial DNA polymerase ␥ (mtDNA Pol ␥) (7-9). However, there are observed discrepancies between toxicity and the potential to inhibit mtDNA Pol ␥, suggesting alternative mechanisms and evaluation of additional host cell polymerases as potential perpetrators of antiviral toxicity (10, 11). Understanding the propensity for host cell polymerases to incorporate nucleoside analogs is critical for assessing safety in the design and development of antiviral, -parasitic, -bacterial, and -cancer nucleoside analog therapies. For instance, development of the antiviral ribonucleoside triphosphate analog BMS-986094 for the treatment of hepatitis C virus was halted in phase II after nine patients were hospitalized and one died (12). Investigational in vitro studies had identified that BMS-986094 was incorporated by the human mitochondrial RNA polymerase 30-fold more ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%