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

Abstract

The human DNA polymerase gamma (Pol c) is responsible for DNA replication in mitochondria. Pol c is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol c-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol c active site almost identically to the substrate dCTP, providing a structural basis for Pol c-mediated drug toxicity. When compared to the apo form, Pol c 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 cB, 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 c mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol c, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol c-mediated drug toxicity.

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ

“…2). This configuration promotes DNA synthesis, as the Pol γA-distal Pol γB contact area allosterically regulates pol and exo activities in the holoenzyme (17).…”

“…The primer strand is terminated by a dideoxynucleotide, and the complexes therefore correspond to inhibitor-bound states. The structures were determined using molecular replacement using a Pol γ ternary complex containing dCTP (17). The statistics for data collection and structural refinement are presented in Table S1.…”

“…X-ray diffraction data were collected at 100 K using synchrotron sources at Advanced Photon Source and Advanced Light Sources and processed with HKL (45). The structures were determined by molecular replacement method using the previously determined Pol γ-DNA-dNTP ternary complex structure, and refined using the program Phenix (17,18). The experimental maps around the (+)-FTC-TP or (-)-FTC-TP binding site are illustrated in Figs.…”

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

“…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). Obtaining ternary X-ray structures of PrimPol in complex with the natural nucleotides and NRTIs will provide structural insights into nucleotide selectivity and PrimPol's incorporation of the relevant NRTIs relative to mtDNA Pol ␥ and HIV RT, facilitating the design of more selective and safe nucleoside analogs (44). Moreover, PrimPol's ability to utilize NTPs indicates that it may also mediate ribonucleoside triphosphate analog toxicities (13) and warrants further investigation.…”

Inherited mitochondrial genomic instability and chemical exposures

“…However, NRTIs not only readily inhibit viral reverse transcriptase, for example, the HIV reverse transcriptase, but they can also inhibit some cellular DNA polymerases (Copeland et al 1992). NRTIs can inhibit DNA polymerases alpha and beta, but the most sensitive to NRTIs is DNA polymerase gamma, the replicative mtDNA polymerase (Kohler and Lewis 2007; Szymanski et al 2015). Inhibition of mtDNA replication leads to mtDNA depletion (Chan and Copeland 2009), and in some cases can cause mtDNA lesions (Chan et al 2007) and oxidative stress (Kline et al 2009).…”