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. 2015 Jul 14;34(14):1959-70.
doi: 10.15252/embj.201591520. Epub 2015 Jun 8.

Structural Basis for Processivity and Antiviral Drug Toxicity in Human Mitochondrial DNA Replicase

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Free PMC article

Structural Basis for Processivity and Antiviral Drug Toxicity in Human Mitochondrial DNA Replicase

Michal R Szymanski et al. EMBO J. .
Free PMC article

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.

Keywords: DNA replication; drug toxicity; human DNA polymerase gamma; mitochondrial toxicity; nucleoside reverse transcriptase inhibitors.

Figures

Figure 1
Figure 1
Structure of Pol γ replication complex

Pol γ holoenzyme ternary complex contains Pol γA, a dimeric Pol γB with the proximal (green) and the distal monomers (blue), a primer/template (pink/gray) DNA duplex, and an incoming nucleotide.

Pol γ (shown in electrostatic surface) interaction with primer/template DNA (green).

Primer/template DNA sequence. The structurally visible region of the primer/template is bolded in the sequences. For convenience, bases are number such that the incoming nucleotide is +1, and the upstream residues are −1, −2, …, n, and the downstream residues +2, +4.

Figure 2
Figure 2
The pol active site of Pol γ ternary complex

The pol active site of a Pol γ ternary complex with a substrate dCTP.

The pol active site of a Pol γ ternary complex with inhibitor ddCTP (zalcitabine).

Figure 3
Figure 3
Schematic representation of Pol γ–DNA interactions Residues are color-coded with exo domain residues in black, spacer domain: IP subdomain in yellow, AID subdomain in orange, pol domain containing subdomains palm (red), fingers (blue) and thumb (green).
Figure 4
Figure 4
Conformational changes induced by DNA binding

The K-tract region moves 25 Å from its position in the apo enzyme (light colors) to the replication complex (solid colors).

The fingers move 20° (10 Å) from the open conformation in apo (light blue) to the closed form in the complex (dark blue).

Figure 5
Figure 5
The inter-subunit conformational changes from apo to ternary complex

Pol γB and its binding elements in Pol γA, the thumb and the L-helix rotate 22° as a rigid body.

The distal Pol γB monomer moves 16 Å from its position in apo to complex position as shown in (C).

The complex.

Figure 6
Figure 6
Pre-steady state kinetics assays of K-tract mutants Assays were performed on the 65-nt DNA template annealed to a 5′-32P-labeled 25-nt primer and initiated by the addition of dNTPs. DNA synthesis products synthesized by Pol γA variants alone (top panels) or holoenzymes (bottom panels).
Figure 7
Figure 7
Regulator for pol and exo activity

The β-hairpin is situated between pol and exo active sties and is connected to the thumb subdomain. R852 interacts with residues on the fingers subdomain.

R853 forms H-bonds with the nascent bp of incoming nucleotide and the template residue (T+1).

Upstream, R853 interacts with bp of the T-1 template and P-1 primer residues.

Figure 8
Figure 8
Structural rationalization of deleterious Pol γ mutations implicated in human diseases

Pol γB G451E mutations illustrated on the structure of the holoenzyme replication complex.

The proximal monomer G451E interacts with the L-helix of Pol γA.

The distal monomer G451E interacts with the Pol γA R232 region.

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