The Common A467T Mutation in the Human Mitochondrial DNA Polymerase (POLG) Compromises Catalytic Efficiency and Interaction with the Accessory Subunit

Abstract

Among the nearly 50 disease mutations in the gene for the catalytic subunit of human DNA polymerase ␥, POLG, the A467T substitution is the most common and has been found in 0.6% of the Belgian population. The A467T mutation is associated with a wide range of mitochondrial disorders, including Alpers syndrome, juvenile spinocerebellar ataxia-epilepsy syndrome, and progressive external ophthalmoplegia, each with vastly different clinical presentations, tissue specificities, and ages of onset. The A467T mutant enzyme possesses only 4% of wild-type DNA polymerase activity, and the catalytic defect is manifest primarily through a 6-fold reduction in k cat with minimal effect on exonuclease function. Human DNA polymerase ␥ (pol ␥) requires association of a 55-kDa accessory subunit for enhanced DNA binding and highly processive DNA synthesis. However, the A467T mutant enzyme failed to interact with and was not stimulated by the accessory subunit, as judged by processivity, heat inactivation, and N-ethylmaleimide protection assays in vitro. Thermolysin digestion and immunoprecipitation experiments further indicate weak association of the subunits for A467T pol ␥. This is the first example of a mutation in POLG that disrupts physical association of the pol ␥ subunits. We propose that reduced polymerase activity and loss of accessory subunit interaction are responsible for the depletion and deletion of mitochondrial DNA observed in patients with this POLG mutation.

Abundance of the POLG disease mutations in Europe, Australia, New Zealand, and the United States explained by single ancient European founders

“…One cause of effective spreading of a recessive allele could be selective advantage for heterozygote carriers, increasing either reproductive fitness or survival of the offspring. Available functional evidence on the consequences of A467T and W748S mutations for POLG, however, does not support selective advantage for them, since the mutant enzymes are catalytically defective, 17,28 and would lead to mtDNA depletion and/or increased mutagenesis. Furthermore, the frequency of the A467T mutation in Australia (Thorburn et al, manuscript in preparation) seems to parallel the observed frequencies in European countries (0.6% in Belgium and 1% in Norway 13,15 ), reflecting the European origin of the Australians.…”

Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication

“…Enzymatic Assays-DNA polymerase activity was determined using the standard pol ␥ assay with poly(dA)-oligo(dT) [12][13][14][15][16][17][18] (Amersham Biosciences) as the primer-template substrate. For steady state kinetic values the same assay was performed in the presence and absence of the p55 accessory subunit using poly(dA)-oligo(dT) [12][13][14][15][16][17][18] as substrate, with reactions containing 25 mM NaCl as previously described (4).…”

“…Mutation of POLG was first linked to Alpers syndrome in 2004 (13), and to date 45 different point mutations in POLG (18 localized to the polymerase domain) are associated with Alpers syndrome (9, 14, 15). However, only two Alpers mutations (A467T and W748S, both in the linker region) have been biochemically characterized (16,17).During the initial cloning and sequencing of the human, Drosophila, and chicken pol ␥ genes, we noted a highly conserved region N-terminal to motif A in the polymerase domain that was specific to pol ␥ (18). This region corresponds to part of the thumb subdomain that tracks DNA into the active site of both Escherichia coli pol I and T7 DNA polymerase (19 -21).…”

“…Mutation of POLG was first linked to Alpers syndrome in 2004 (13), and to date 45 different point mutations in POLG (18 localized to the polymerase domain) are associated with Alpers syndrome (9, 14, 15). However, only two Alpers mutations (A467T and W748S, both in the linker region) have been biochemically characterized (16,17).…”

Quantitative multiplex PCR of short fluorescent fragments for the detection of large intragenic POLG rearrangements in a large French cohort

“…Although no clear genotype-phenotype correlation could be established in patients with POLG mutations, both patients had a severe phenotype with epilepsy and mtDNA depletion in liver (8% and 30%, respectively). Large deletions associated with other mutations resulting in very low wild-type DNA polymerase activity, such as p.Ala467Thr or p.Trp748Ser, 32,33 could lead to severe cases of childhood disease.…”