A mechanistic view of human mitochondrial DNA polymerase γ: Providing insight into drug toxicity and mitochondrial disease

Bailey, Christopher M.; Anderson, Karen S.

doi:10.1016/j.bbapap.2010.01.007

Cited by 24 publications

(15 citation statements)

References 114 publications

(152 reference statements)

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“…NRTI incorporation as catalyzed by PrimPol. Previous work in our lab has evaluated the kinetic mechanism and NRTI-associated toxicity in the mitochondria using detailed mechanistic studies to examine incorporation of NRTIs by HIV RT and mtDNA Pol ␥ (9,(28)(29)(30)(31)(32)(33)(34)(35). With the recent discovery of PrimPol in the mitochondria (15), an obvious question is whether additional mechanisms for NRTI-mediated toxicity may be plausible.…”

Section: Resultsmentioning

confidence: 99%

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%

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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“…This is the case in particular when external factors change the balance between the amount of mtDNA, and the protein levels of TFAM. For example, some anti-viral drugs such as AZT and ddC inhibit mtDNA replication, such that the ratio of TFAM: mtDNA is increased transiently [90]. Here, we would propose that Lon monitors and adjusts the TFAM: mtDNA ratio to sustain appropriate mtDNA transcription levels by selective degradation of TFAM (Fig.…”

Section: Potential Physiological Roles Of Matrix Aaa+ Proteases Inmentioning

confidence: 99%

Matrix proteases in mitochondrial DNA function

Matsushima

Kaguni

2012

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Lon, ClpXP and m-AAA are the three major ATP-dependent proteases in the mitochondrial matrix. All three are involved in general quality control by degrading damaged or abnormal proteins. In addition to this role, they are predicted to serve roles in mitochondrial DNA functions including packaging and stability, replication, transcription and translation. In particular, Lon has been implicated in mtDNA metabolism in yeast, fly and humans. Here, we review the role of Lon protease in mitochondrial DNA functions, and discuss a putative physiological role for mitochondrial transcription factor A (TFAM) degradation by Lon protease. We also discuss the possible roles of m-AAA and ClpXP in mitochondrial DNA functions, and the putative candidate substrates for the three matrix proteases.

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“…The structure of mtDNA polγ has recently become available to correlate with functional studies [39, 40]. Mechanistic aspects of mtDNA pol γ and its importance in mitochondrial disease, have recently been reviewed [41]. …”

Section: Transient Kinetics To Investigate the Mechanism Of Rt Andmentioning

confidence: 99%

“…Steady-state [42] and transient kinetic studies have been used to examine the incorporation efficiency of the triphosphate form of these nucleoside analogs by RT and mtDNA Pol γ [41, 43, 44]. Knowledge of the incorporation efficiency coupled with an assessment of the proofreading exonuclease activity for the mtDNA polymerases for removal of NRTIs has been used to develop a toxicity index that allows us to predict NRTI toxicity at the level of the triphosphate.…”

Section: Nucleoside Analogs As Inhibitors Of Dna Polymerization: Mmentioning

confidence: 99%

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A transient kinetic approach to investigate nucleoside inhibitors of mitochondrial DNA polymerase γ

Anderson

2010

Methods

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Nucleoside analogs play an essential role in treating human immunodeficiency virus (HIV) infection since the beginning of the AIDS epidemic and work by inhibition of HIV-1 reverse transcriptase (RT), a viral polymerase essential for DNA replication. Today, over 90% of all regimens for HIV treatment contain at least one nucleoside. Long-term use of nucleoside analogs has been associated with adverse effects including mitochondrial toxicity due to inhibition of the mitochondrial polymerase, DNA polymerase gamma (mtDNA pol ©). In this review, we describe our efforts to delineate the molecular mechanism of nucleoside inhibition of HIV-1 RT and mtDNA pol © based upon a transient kinetic approach using rapid chemical quench methodology. Using transient kinetic methods, the maximum rate of polymerization (k pol ), the dissociation constant for the ground state binding (K d ), and the incorporation efficiency (k pol /K d ) can be determined for the nucleoside analogs and their natural substrates. This analysis allowed us to develop an understanding of the structure activity relationships that allow correlation between the structural and stereochemical features of the nucleoside analog drugs with their mechanistic behavior toward the viral polymerase, RT, and the host cell polymerase, mtDNA pol γ. An indepth understanding of the mechanisms of inhibition of these enzymes is imperative in overcoming problems associated with toxicity.

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A mechanistic view of human mitochondrial DNA polymerase γ: Providing insight into drug toxicity and mitochondrial disease

Cited by 24 publications

References 114 publications

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

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

Matrix proteases in mitochondrial DNA function

A transient kinetic approach to investigate nucleoside inhibitors of mitochondrial DNA polymerase γ

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