Disease mutations in the human mitochondrial DNA polymerase thumb subdomain impart severe defects in mitochondrial DNA replication

Abstract

Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol gamma), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol gamma revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g-->a; T851A, c.2551a-->g; R852C, c.2554c-->t; R853Q, c.2558g-->a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g-->t and T885S, c.2653a-->t). Biochemical characterization of purified, recombinant forms of pol gamma revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50-70% wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol gamma accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol gamma and examines the consequences of mitochondrial disease mutations in this region.

FIGURE 1.

POLG mutations characterized in this study. A, the location of the six mutations characterized is shown in red in the primary sequence of pol γ. Four mutations, the G848S, T851A, R852C, and R853Q, are located in the thumb domain, whereas two mutations, the Q879H and T885S, are in the palm domain of the polymerase region. B, sequence alignment of pol γ from yeast to humans. The amino acids characterized in this study are shown in red. Yellow-highlighted amino acids are highly conserved, and blue-highlighted amino acids are moderately conserved.

FIGURE 2.

Secondary structure of Alpers syndrome mutant p140 proteins and their stability. A, CD spectroscopy of WT and mutant p140 proteins (WT (red circles), G848S (violet circles), T851A (yellow circles), R852C (black circles), R853Q (blue circles), Q879H (orange circles), and T885S (brown circles)). B, thermal stability of WT and mutant p140 proteins. The enthalpy change during protein folding (ΔH_m), measured at 220 nm, is represented by bars with S.D. The melting temperature (T_m) is represented by squares. The results are an average of three independent experiments.

FIGURE 3.

Polymerase activity of Alpers syndrome mutant p140 proteins. Enzyme activities of the WT and p140 mutants were measured using poly(dA)-oligo(dT)_12–18 substrate (A) and activated calf-thymus DNA substrate (B) as described under “Experimental Procedures.” The specific enzymatic activity of each protein is represented as units/ng of the p140 protein, where 1 unit is defined as the number of pmol of dTTP incorporated per ng of the p140 protein per h at 37 °C. The average result of three experiments is shown with S.D.

FIGURE 4.

DNA binding affinity of Alpers mutants. A, electrophoretic mobility shift assays were performed using WT and mutant p140 enzymes as described under “Experimental Procedures” to estimate the K_d(DNA) values. The average result of three experiments is shown with S.D. B, prediction of the location of amino acids in the α-helix of the thumb domain with residue numbers and positions labeled. Prediction was generated using the helical wheel drawing program. (Rutgers, Bioinformatics Laboratory).

FIGURE 5.

Immunoblot showing physical association of Alpers syndrome mutant p140 proteins with the p55 accessory subunit. Co-immunoprecipitation assays were performed using WT and mutant p140 proteins with immobilized anti-p55 rabbit antibodies as described under “Experimental Procedures.” Lane 1, 250 ng of purified p140 and p55 loaded directly for positive control; lane 2, WT p140 alone; lane 3, p55 alone; lane 4, WT p140 and p55; lane 5, G848S p140 and p55; lane 6, T851A p140 and p55; lane 7, R852C p140 and p55; lane 8, R853Q p140 and p55; lane 9, Q879H p140 and p55; lane 10, T885S p140 and p55.

FIGURE 6.

Functional interaction of Alpers syndrome mutant p140 proteins with the p55 accessory subunit. Primer extension assays were performed using WT and mutant p140 enzymes in the presence and absence of p55 accessory subunit on singly primed M13 DNA as described under “Experimental Procedures.” Reactions contained 20 fmol of substrate (all lanes), 50 fmol of p140 enzymes (lanes 3–6, WT; lane 7–10, G848S; lanes 11–14, T851A; lanes 15–18, R852C; lanes 19–22, R853Q; lanes 23–26, Q879H; lanes 27–30, T885S), 100 fmol of p55 accessory subunit (lanes 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, and 30). Activity was measured at 0 mm NaCl (odd-numbered lanes) and at 75 mm NaCl (even-numbered lanes). Lanes 1 and 2 had no enzyme. The arrow indicates the position of the unextended 35-mer primer.