Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase, Pol γ

Abstract

Mutations in Pol γ represent a major cause of human mitochondrial diseases, especially those affecting the nervous system in adults and in children. Recessive mutations in Pol γ represent nearly half of those reported to date, and they are nearly uniformly distributed along the length of the POLG1 gene (Human DNA Polymerase gamma Mutation Database); the majority of them are linked to the most severe form of POLG syndrome, Alpers-Huttenlocher syndrome. In this report, we assess the structure-function relationships for recessive disease mutations by reviewing existing biochemical data on site-directed mutagenesis of the human, Drosophila and yeast Pol γs, and their homologs from the family A DNA polymerase group. We do so in the context of a molecular model of Pol γ in complex with primer-template DNA, which we have developed based upon the recently solved crystal structure of the apoenzyme form. We present evidence that recessive mutations cluster within five distinct functional modules in the catalytic core of Pol γ. Our results suggest that cluster prediction can be used as a diagnosis-supporting tool to evaluate the pathogenic role of new Pol γ variants.

Figure 1.

Alpers disease mutations cluster within functional modules in the catalytic subunit of Pol γ. Upper panel: schematic diagram of the POLG1 gene showing the distribution of recessive Alpers disease mutations (Human DNA Polymerase gamma Database, http://tools.niehs.nih.gov/polg/). AID and IP in the spacer domain refer to the accessory (subunit) interacting and intrinsic processivity subdomains, respectively, that are discussed in the text; NTD refers to the N-terminal domain. Lower panel: tertiary structural representation of the apoenzyme form of Pol γ [PDB code 3IKM, (7)] with modeled DNA, identifying the positions of five functional modules (shown in mesh) that are defined by clusters of amino acid residues (shown as spheres) affected by Alpers disease mutations as follows: Cluster 1, green; Cluster 2, yellow; Cluster 3, red; Cluster 4, blue; Cluster 5, cyan. The domains of Pol γA are shown as surface representations, and in part as secondary structural elements (SSEs) that are colored as depicted in A. The proximal and distal accessory subunits are shown as surface representations in light and dark gray, respectively. Primer–template DNA was docked as described in the text and is displayed as orange ribbons.

Figure 2.

Alpers Cluster 1 mutations affect the 5′–3′ DNA polymerase activity of Pol γ. Amino acid residues affected by Alpers Cluster 1 mutations in POLG1 are shown as green spheres. Other Pol γ residues that are discussed in the text are shown in brown, and T7 residues are shown in red. Pol domain SSEs are shown in pink according to the schematic shown in Figure 1; ptDNA is indicated by orange (template) and brown (primer) strands. The incoming dNTP is shown in blue. Mg²⁺ ions are shown as small gray spheres. A, upper panel, overview of the positions of Alpers Cluster 1 mutations with dashed black lines indicating the regions described in the text and depicted in B–D; A, lower panel, overview of the positions of Alpers Cluster 1 mutations relative to ptDNA and incoming dNTP; B–D, positioning of Pol γ residues in the apoenzyme form [PDB code 3IKM (7)] and T7 Pol residues in its closed ternary complex [PDB code 1T8E (25)] relative to ptDNA and incoming dNTP, with the dashed arrow showing the expected movements of the Pol γ residues upon formation of a closed complex: B, Alpers mutations surrounding the Mg²⁺-binding residues; C, Alpers mutations affecting O-helix movement and dNTP binding, with the T7 Pol O-helix in its closed conformation superimposed in red; D, Alpers mutations affecting the RR loop and the surrounding residues.

Figure 3.

Alpers Cluster 2 mutations affect the upstream DNA-binding channel of Pol γ. Amino acid residues affected by Alpers Cluster 2 mutations in POLG1 are shown as yellow spheres. Other Pol γ residues that are discussed in the text are shown in brown. Spacer domain SSEs are shown in magenta and pol domain SSEs are in pink according to the schematic shown in Figure 1; ptDNA is indicated by orange (template) and brown (primer) strands. The spacer domain is also shown as a transparent surface representation in pale gray and the exo domain is shown in purple.

Figure 4.

Alpers Cluster 3 mutations are associated with a novel Pol γ-specific functional module proposed to be involved in primer strand partitioning between the pol and exo sites. Amino acid residues affected by Alpers Cluster 3 mutations in POLG1 are shown as red spheres/mesh adjacent to a novel alpha helix with an associated loop–hairpin (the partitioning loop), also shown in red. The brown spheres and mesh represent the SYW (fly)/ SFW (human) and surrounding residues, respectively, that are described in the text. The pol domain is shown as a surface representation in pink and the exo domain is shown in purple, according to the schematic shown in Figure 1; ptDNA is indicated by orange (template) and brown (primer) strands. (A) The predicted position of the partitioning loop relative to ptDNA in the pol mode, and (B) represents the exo mode. To dock the frayed ptDNA in the exo active site, the exo domain residues 324–518 of Klenow (PDB code 1KLN) were aligned with the exo domain residues 170–440 of Pol γ (PDB code 3IKM) (Supplementary Figure S3).

Figure 5.

Alpers Cluster 4 mutations affect Pol γA interactions with the distal Pol γB upon DNA binding by Pol γ holoenzyme. Amino acid residues affected by Alpers Cluster 4 mutations in POLG1 are shown as blue spheres/ mesh and are located largely within the exo domain (shown in purple). The helix shown in magenta is the AID (accessory interacting domain) of the spacer region that interacts with the proximal accessory subunit. Other domains are colored according to the schematic shown in Figure 1; duplex DNA is indicated by the orange strand. Orange spheres indicate the positions of the accessory subunit residues described in the text. The proximal and distal accessory subunits are shown as surface representations in light and dark gray, respectively.

Figure 6.

Alpers Cluster 5 mutations are proposed to affect replisome interactions. Amino acid residues affected by Alpers Cluster 5 mutations in POLG1 are shown as cyan spheres. Spacer domain SSEs within the IP (intrinsic processivity subdomain) are shown in magenta; ptDNA is indicated by orange (template) and brown (primer) strands. The spacer domain is also shown as a transparent surface representation in pale gray and the exo domain is shown in purple. Brown spheres indicate the positions of the IP residues described in the text.

Figure 7.

Combinations of mutations found in Alpers patients. Individual Alpers mutations are grouped by cluster as shown in Figure 1, and black blocks represent Alpers manifesting combinations. The inset in the lower right presents a simplified version of the table by reducing the axes to the five clusters only, where gray blocks represent cluster combinations that have not been found in Alpers patients. The tabulated data suggest that two mutations from the same cluster do not typically manifest as early-onset Alpers disease. Furthermore, the data indicate that Cluster 4 (blue) mutations manifest as Alpers disease only when in combination with Cluster 2 (yellow) or Cluster 5 (cyan) mutations. These trends support the existence of unique functional relationships in Pol γA that are inherent to each cluster.