Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase

Michal R. Szymanski, Vladmir B. Kuznetsov, Christie Shumate, Qingchao Meng, Young Sam Lee, Gayatri Patel, Smita Patel, Yuhui Yin

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

The human DNA polymerase gamma (Pol γ) is responsible for DNA replication in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol γ-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol γ active site almost identically to the substrate dCTP, providing a structural basis for Pol γ-mediated drug toxicity. When compared to the apo form, Pol γ undergoes intra- and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol γB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol γ mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity. Synopsis Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity. Zalcitabine binds to the Pol γ active site in a similar manner as the substrate dCTP. The human mitochondrial DNA polymerase ternary complex halted by zalcitabine provides a structural mechanism for antiviral drug toxicity. The crystal structure suggests the accessory subunit Pol γB to allosterically regulate processivity and proofreading of Pol γ. This study sets the stage for understanding human diseases associated with the mitochondrial DNA polymerase. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity.

Original languageEnglish (US)
Pages (from-to)1959-1970
Number of pages12
JournalEMBO Journal
Volume34
Issue number14
DOIs
StatePublished - 2015

Fingerprint

Zalcitabine
Drug-Related Side Effects and Adverse Reactions
Mitochondrial DNA
Antiviral Agents
Toxicity
DNA-Directed DNA Polymerase
Crystal structure
Substrates
Holoenzymes
DNA Primers
DNA
Accessories
Pharmaceutical Preparations
Catalytic Domain
Dideoxynucleosides
HIV Reverse Transcriptase
Mitochondria
Virus Diseases
DNA replicase
DNA Replication

Keywords

  • DNA replication
  • drug toxicity
  • human DNA polymerase gamma
  • mitochondrial toxicity
  • nucleoside reverse transcriptase inhibitors

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Szymanski, M. R., Kuznetsov, V. B., Shumate, C., Meng, Q., Lee, Y. S., Patel, G., ... Yin, Y. (2015). Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase. EMBO Journal, 34(14), 1959-1970. https://doi.org/10.15252/embj.201591520

Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase. / Szymanski, Michal R.; Kuznetsov, Vladmir B.; Shumate, Christie; Meng, Qingchao; Lee, Young Sam; Patel, Gayatri; Patel, Smita; Yin, Yuhui.

In: EMBO Journal, Vol. 34, No. 14, 2015, p. 1959-1970.

Research output: Contribution to journalArticle

Szymanski, MR, Kuznetsov, VB, Shumate, C, Meng, Q, Lee, YS, Patel, G, Patel, S & Yin, Y 2015, 'Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase', EMBO Journal, vol. 34, no. 14, pp. 1959-1970. https://doi.org/10.15252/embj.201591520
Szymanski, Michal R. ; Kuznetsov, Vladmir B. ; Shumate, Christie ; Meng, Qingchao ; Lee, Young Sam ; Patel, Gayatri ; Patel, Smita ; Yin, Yuhui. / Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase. In: EMBO Journal. 2015 ; Vol. 34, No. 14. pp. 1959-1970.
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abstract = "The human DNA polymerase gamma (Pol γ) is responsible for DNA replication in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol γ-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol γ active site almost identically to the substrate dCTP, providing a structural basis for Pol γ-mediated drug toxicity. When compared to the apo form, Pol γ undergoes intra- and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol γB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol γ mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity. Synopsis Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity. Zalcitabine binds to the Pol γ active site in a similar manner as the substrate dCTP. The human mitochondrial DNA polymerase ternary complex halted by zalcitabine provides a structural mechanism for antiviral drug toxicity. The crystal structure suggests the accessory subunit Pol γB to allosterically regulate processivity and proofreading of Pol γ. This study sets the stage for understanding human diseases associated with the mitochondrial DNA polymerase. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity.",
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N2 - The human DNA polymerase gamma (Pol γ) is responsible for DNA replication in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol γ-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol γ active site almost identically to the substrate dCTP, providing a structural basis for Pol γ-mediated drug toxicity. When compared to the apo form, Pol γ undergoes intra- and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol γB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol γ mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity. Synopsis Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity. Zalcitabine binds to the Pol γ active site in a similar manner as the substrate dCTP. The human mitochondrial DNA polymerase ternary complex halted by zalcitabine provides a structural mechanism for antiviral drug toxicity. The crystal structure suggests the accessory subunit Pol γB to allosterically regulate processivity and proofreading of Pol γ. This study sets the stage for understanding human diseases associated with the mitochondrial DNA polymerase. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity.

AB - The human DNA polymerase gamma (Pol γ) is responsible for DNA replication in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol γ-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol γ active site almost identically to the substrate dCTP, providing a structural basis for Pol γ-mediated drug toxicity. When compared to the apo form, Pol γ undergoes intra- and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol γB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol γ mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity. Synopsis Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity. Zalcitabine binds to the Pol γ active site in a similar manner as the substrate dCTP. The human mitochondrial DNA polymerase ternary complex halted by zalcitabine provides a structural mechanism for antiviral drug toxicity. The crystal structure suggests the accessory subunit Pol γB to allosterically regulate processivity and proofreading of Pol γ. This study sets the stage for understanding human diseases associated with the mitochondrial DNA polymerase. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity.

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