Effect of the Y955C Mutation on Mitochondrial DNA Polymerase Nucleotide Incorporation Efficiency and Fidelity

Abstract

The human mitochondrial DNA polymerase (pol γ) is responsible for the replication of the mitochondrial genome. Mutation Y955C in the active site of pol γ results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson’s disease. In single turnover kinetic studies, we show that the Y955C mutation resulted in a decrease in the maximum rate of polymerization and an increase in the Km for correct incorporation. The mutation decreased the specificity constant for correct incorporation of dGTP, TTP, and ATP to values of 1.5, 0.35, and 0.044 μM−1s−1, respectively, representing reductions of 30-, 110- and 1300-fold relative to wild-type enzyme. The fidelity of incorporation was reduced 6- to 130-fold; largely due the significant decrease in the specificity constant for correct dATP:T incorporation. For example, kcat/Km for forming a TTP:T mismatch was decreased tenfold from 0.0002 to 0.00002 μM−1s−1 by the Y955C mutant, but the 1300-fold slower incorporation of the correct dATP:T relative to wild-type led to a 130-fold lower fidelity. While correct incorporation of 8-oxo-dGTP was largely unchanged, incorporation of 8-oxo-dG with dA in the template strand was reduced 500-fold. These results support a role of Y955 in stabilizing A:T base pairs at the active site of pol γ and suggest that the severe clinical symptoms of patients with this mutation may be due, in part, to the reduced efficiency of dATP incorporation opposite T, and that the autosomal dominant phenotype may arise from the resulting higher mutation frequency.

Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression

“…exo Ϫ cells mapped mutations at nucleotide position 1951 (A to T, A to G, A to C, and a single G insertion between 1950 and 1951). The Pol-␥ showed an average discrimination factor of 3 ϫ 10 5 against mismatched nucleotides, which equals a mutation rate of 3.3 ϫ 10 Ϫ6 /nucleotide/genome replication in the absence of the proofreading activity (8,9). In accordance with in vitro measurements, the mutation rate of exo Ϫ cells after ϳ4 -5 generations is 1.7 ϫ 10 Ϫ5 (Table 1).…”

“…Nucleotide Incorporation Assays-Single nucleotide incorporation assays were performed with a RQF-3 rapid quench flow apparatus (KinTek Corp.) as described (9). In brief, 100 nM Pol-␥ exo ϩ enzyme was preincubated with 50 nM 5Ј-32 P-labeled DNA template (25-mer/45-mer duplex) on ice for 10 min, and the complex was rapidly mixed with 12.5 mM Mg 2ϩ and 50 M dATP for variable time at 37°C before the reaction was quenched by mixing with 250 mM EDTA.…”

“…Kinetic analysis of purified Pol-␥ mutants showed a ϳ150-, 11-, and 1300-fold decrease in the rate of dATP incorporation for H932Y, Y951N, and Y955C, respectively, compared with the wild-type enzyme (1,8,9). 4 Strikingly, the fidelity of Pol-␥ mutants as calculated from the ratio of k cat /K m values for correct (dATP:dT) versus mismatched (TTP: dT) base pairs dropped ϳ100-fold for Y955C and ϳ3-fold for H932Y (8,9).…”

“…Another active site mutation in Pol-␥, Y955C, was found in patients presenting "early onset autosomal dominant PEO" in their twenties (16). Kinetic analysis showed a 1300-fold reduction in k cat /K m while incorporating a dATP base opposite a template T (9,11).…”

DNA Polymerase Beta Participates in Mitochondrial DNA Repair

“…Often the severity of the disease is not predicted by the amount of residual Pol␥ activity. Particularly perplexing is the Y955C mutation in POLGA, the most common and severe autosomal dominant mutation causing CPEO and Parkinsonism (61). The mutation causes Pol␥ incorporation levels to drop below 1% of control activity, yet the life span of these patients is measured not in days or weeks but rather in decades, and the individuals are often symptomless well into adulthood.…”