A mechanistic view of human mitochondrial DNA polymerase gamma: providing insight into drug toxicity and mitochondrial disease

Abstract

Mitochondrial DNA polymerase gamma (Pol gamma) is the sole polymerase responsible for replication of the mitochondrial genome. The study of human Pol gamma is of key importance to clinically relevant issues such as nucleoside analog toxicity and mitochondrial disorders such as progressive external ophthalmoplegia. The development of a recombinant form of the human Pol gamma holoenzyme provided an essential tool in understanding the mechanism of these clinically relevant phenomena using kinetic methodologies. This review will provide a brief history on the discovery and characterization of human mitochondrial DNA polymerase gamma, focusing on kinetic analyses of the polymerase and mechanistic data illustrating structure-function relationships to explain drug toxicity and mitochondrial disease.

Figure 1. Proposed models of mitochondrial genome replication

Schematic representation of strand displacement, strand-coupled and RITOLS replication of the mitochondrial genome. (Reprinted from Trends in Biochemical Sciences, Vol 34 / issue 7, Ian J. Holt, Mitochondrial DNA replication and repair: all a flap, Pages 358-365, Copyright 2009, with permission from Elsevier)

Figure 2. Crystal structure of Pol γ holoenzyme

Image depicts the crystal structure of the heterotrimeric Pol γ holoenzyme [42]. Fingers (orange), palm (green) and thumb (blue) subdomains of the canonical right hand organization of the polymerase domain are shown. Additional domains highlighted include the mitochondrial localization sequence (yellow), exonuclease (red) and spacer subdomain (purple). The proximal (cyan) and distal (light cyan) monomers of the dimeric accessory subunit are shown.

Figure 3. Kinetic Scheme for Pol γ Polymerization

Schematic demonstrating rate constants for dNTP incorporation and removal by Pol γ as determined by pre-steady state kinetic analysis. Figure adapted from [49].

Figure 4. Structures of selected clinically relevant nucleoside analogs

Figure 5. Molecular basis for Pol γ sensitivity to ddNTP inhibition

A) Pol I is resistant to dideoxynucleotide inhibition, which has been linked to the presence of a phenyalanine at position 667 on the O-helix, shown in fuschia. Image created from the ternary complex of the catalytic subunit ternary complex of Klentaq [120]. B) Molecular model of the Pol γ active site [112] demonstrates the phenolic hydroxyl group of Y951 mimicking the 3′-OH of a bound dNTP, allowing efficient incorporation of ddNTPs. ddCTP is shown bound to the Pol I and Pol γ active sites in green, with coordinated Mg²⁺ ions (gray), and catalytic residues shown in stick form.

Figure 6. Molecular Model of Pol γ Active Site

Several residues mutated in PEO and discussed in the text are shown in cyan. NRTI susceptibility is linked to mutation of R964, shown in hot pink. ddCTP is shown bound to the active site in grey. Molecular model was created using the ternary structure of T7 polymerase and was previously described [112].