Role of histidine 932 of the human mitochondrial DNA polymerase in nucleotide discrimination and inherited disease

Abstract

The human mitochondrial DNA polymerase (pol γ) is nuclearly encoded and is solely responsible for the replication and repair of the mitochondrial genome. The progressive accumulation of mutations within the mitochondrial genome is thought to be related to aging, and mutations in the pol γ gene are responsible for numerous heritable disorders including progressive external opthalmoplegia, Alpers syndrome, and parkinsonism. Here we investigate the kinetic effect of H932Y, a mutation associated with opthalmoplegia. Mutations H932Y and H932A reduce the specificity constant governing correct nucleotide incorporation 150- and 70-fold, respectively, without significantly affecting fidelity of incorporation or the maximum rate of incorporation. However, this leads to only a 2-fold reduction in rate of incorporation at a physiological nucleotide concentration (∼100 μm). Surprisingly, incorporation of T:T or C:T mismatches catalyzed by either H932Y or H932A mutants was followed by slow pyrophosphate release (or fast pyrophosphate rebinding). Also, H932Y readily catalyzed incorporation of multiple mismatches, which may have a profound physiological impact over time. His-932 is thought to contact the β-phosphate of the incoming nucleotide, so it is perhaps surprising that H932Y appears to slow rather than accelerate pyrophosphate release.

FIGURE 1.

Catalytic residues on the fingers domain. Residues Lys-947, His-932, and Arg-943 provide hydrogen bonds to the α-, β-, and γ-phosphates of the incoming dNTP. In this figure the structure of T7 DNA polymerase, 1T7P.pdb, (5) was used to model the locations of the homologous residues in pol γ. The template strand is blue, the primer strand is green, the incoming dNTP is shown in magenta, and the catalytic residues are in yellow. Template positions are labeled relative to the templating nucleotide (T0).

FIGURE 2.

Kinetics of incorporation of dATP and ddATP for the WT and H932Y mutant. For each concentration series, a preformed enzyme-DNA complex ([enzyme] > [DNA duplex]) was rapidly mixed with Mg²⁺ and various concentrations of dATP or ddATP. In each experiment the final concentrations of the enzyme and DNA after mixing were 150–175 and 75–100 nm, respectively. In globally fitting each data set, the concentration of active enzyme was adjusted to fit the amplitude. A, incorporation of dATP for WT exo⁻ pol γ at various concentrations (0.2 (●), 0.5 (○), 1.5 (■), 3 (□), 5.5 (▴), 8.5 (△) μm) was globally fit to Scheme 1 yielding a k_pol of 30 ± 2 s⁻¹ and a K_d,app of 0.7 ± 0.14 μm. B, incorporation of dATP for H932Y exo⁻ pol γ at various concentrations (2.5 (●), 7.5 (○), 20 (■), 40 (□), 100 (▴), 500 (△) μm from bottom to top) was globally fit to Scheme 1 yielding a k_pol of 28.6 ± 2.9 s⁻¹ and a K_d,app of 103 ± 15 μm. C, incorporation of ddATP for WT exo⁻ pol γ at various concentrations (0.01 (●), 0.025 (○), 0.05 (■), 0.1 (□), and 5 (▴) μm) was globally fit to Scheme 1 yielding a k_pol of 1.8 ± 0.2 s⁻¹ and a K_d,app of 0.42 ± 0.06 μm. D, incorporation of ddATP for H932Y exo⁻ polγ at various concentrations (0.5 (●), 2 (○), 5 (■), 15 (□), and 500 (▴) μm) was globally fit to Scheme 1 yielding a k_pol of 1 ± 0.14 s⁻¹ and a K_d,app of 61 ± 10 μm.

FIGURE 3.

Kinetics of misincorporation by WT enzyme and H932Y mutant. For each concentration dependence, a preformed enzyme-DNA complex ([enzyme] > [DNA duplex]) was rapidly mixed with Mg²⁺ and various concentrations of incorrect nucleotide. The time course of product formation was then fit globally. In each experiment, the final concentrations of the enzyme and DNA after mixing were 100–150 and 75 nm, respectively. In globally fitting each data set, the concentration of active enzyme was adjusted to fit the amplitude. A, formation of a T:T mismatch by WT exo⁻ pol γ at each TTP concentration (1.5 (●), 5 (○), 15 (■), 50 (□), and 250 (▴) μm) was globally fit to the mechanism shown in Scheme 1, yielding an apparent K_d of 81.8 ± 10.9 μm and k_pol of 0.01 ± 0.004 s⁻¹. B, formation of a C:T mismatch by WT exo⁻ pol γ at each dCTP concentration (15 (●), 50 (○), 250 (■), and 1000 (□) μm) was fit globally to the mechanism shown in Scheme 1, yielding an apparent K_d of 1030 ± 193 μm and k_pol of 0.06 ± 0.018 s⁻¹. C, formation of a T:T mismatch by H932Y exo⁻ mutant at each TTP concentration (15 (●), 50 (○), 125 (■), 250 (□), 1000 (▴), and 5000 (△) μm) was fit globally (solid line) to the mechanism shown in Scheme 2, yielding an apparent K_d of 1630 ± 310 μm, k₂ of 0.1 ± 0.02 s⁻¹, k₋₂ of 0.01 ± 0.003 s⁻¹, and k₃ of 0.0004 ± 0.0005 s⁻¹. The dashed line indicates an attempt to fit the data with the mechanism shown in Scheme 1, showing that it cannot account for the concentration dependence of the rate and amplitude. D, formation of a C:T mismatch by H932Y exo⁻ mutant at various dCTP concentrations (175 (●), 375 (○), 750 (■), 1500 (□), and 4000 (▴) μm) were globally fit to the mechanism shown in Scheme 2, yielding an apparent K_d of 22200 ± 3420 μm, k₂ of 0.02 ± 0.005 s⁻¹, k₋₂ of 0.0004 ± 0.003 s⁻¹, and k₃ of 0.0003 ± 0.002 s⁻¹. Traces for the formation of a G:T mismatch are not shown, but all time courses were fit to the mechanism shown in Scheme 1, and the resulting parameters are summarized in Table 2.

FIGURE 4.

Misincorporation of TTP by H932Y exo⁻ pol γ. Products from the incorporation of TTP (at 1000 μm) were resolved on a 15% polyacrylamide sequencing gel and revealed multiple incorporations past a T:T mismatch. The left axis displays the mismatch formed according to the templating base of the DNA substrate corresponding to each band. Above the figure we show the DNA sequence used. The details of the experiment and the analysis and fitting of these data are presented in Fig. 3B.

FIGURE 5.

Confidence contours for the global fit to four rate constants showing all six pairwise combinations of the four rate constants. The results were derived by globally fitting the data collected as shown in Fig. 3B. The central red zone shows the area of good fit, and the yellow band between the red and green zones shows the threshold representing a 10% increase in χ², which is used to set upper and lower confidence limits on each of the kinetic parameters (21).

FIGURE 6.

Kinetics of incorporation of for the H932A mutant. For each concentration series, a preformed enzyme-DNA complex ([enzyme] > [DNA duplex]) was rapidly mixed with Mg²⁺ and various concentrations of nucleotide. The time course of product formation was then fit globally (solid lines). In each experiment the final concentrations of the enzyme and DNA after mixing were 100–175 and 75–100 nm, respectively. In globally fitting each data set, the concentration of active enzyme was adjusted to fit the amplitude. A, incorporation of dATP for H932A exo⁻ pol γ at various concentrations (0.2 (●), 0.5 (○), 2 (■), 4 (□), 7 (▴), 11 (△), 20 (▾), 30 (▿), and 55 (♦) μm) was globally fit to Scheme 1 yielding a k_pol of 23 ± 2.8 s⁻¹ and a K_d,app of 39 ± 6.4 μm. B, incorporation of ddATP for H932A exo⁻ pol γ at various concentrations (0.5 (●), 2 (○), 5 (■), 15 (□), and 500 (▴) μm) was globally fit to Scheme 1 yielding a k_pol of 0.7 ± 0.04 s⁻¹ and a K_d,app of 38 ± 2.6 μm. C, formation of a T:T mismatch by H932A exo⁻ pol γ at each TTP concentration (15 (●), 50 (○), 125 (■), 250 (□), and 5000 (▴) μm) was globally fit to the mechanism shown in Scheme 2, yielding an apparent K_d of 9400 ± 2700 μm, k₂ of 0.47 ± 0.15 s⁻¹, a k₋₂ of 0.013 ± 0.0056 s⁻¹, and a k₃ of 0.0003 ± 0.0004 s⁻¹. D, formation of a C:T mismatch by H932A exo⁻ pol γ at each dCTP concentration (175 (●), 375 (○), 750 (■), 1500 (□), and 5000 (▴) μm) was also globally fit to the mechanism shown in Scheme 2, yielding an apparent K_d of 40900 ± 9470 μm, k₂ of 0.038 ± 0.012 s⁻¹, k₋₂ of 0.0009 ± 0.0006 s⁻¹, and k₃ of 0.0005 ± 0.001. Traces for the formation of a G:T mismatch are not shown, but all time courses were fit to the mechanism shown in Scheme 1, and the resulting parameters are summarized in Tables 2 and 3.