Disease variants of the human mitochondrial DNA helicase encoded by C10orf2 differentially alter protein stability, nucleotide hydrolysis, and helicase activity

Abstract

Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg(2+) ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.

FIGURE 1.

Schematic of the human C10orf2 gene shows the positions of missense mutations associated with disease. The five exons of C10orf2 are preceded by three in-frame stop codons (dots) (5). The positions of the amino-terminal mitochondrial targeting sequence (amino acids 1–42) and the conserved motifs of superfamily 4 DNA helicases are shown. Missense mutations associated with PEO (plain text) and MDS (underlined) are indicated (5, 14, 44, 45, 47, 51–62). The Y508C substitution is also associated with IOSCA (43, 44, 46, 47).

FIGURE 2.

Purification of recombinant human mitochondrial DNA helicase from E. coli. A, samples of affinity-tagged p72 were taken at each stage of purification and analyzed by SDS-PAGE on a 4–20% gradient gel. Proteins were stained with Coomassie Brilliant Blue. Lane 1, crude cell lysate after induction; lane 2, soluble lysate; lane 3, IMAC unbound fraction; lane 4, IMAC wash step; lane 5, IMAC-bound fraction; lane 6, heparin unbound fraction; lane 7, HiTrap Q-bound fraction; lanes 8 and 9, heparin peak fractions. The positions of molecular mass markers (kDa) are indicated. The arrow shows the position of the recombinant 72-kDa protein. B, samples of the indicated fractions were analyzed by electrophoresis through 1% agarose gels in buffer containing 40 mm Tris base, 20 mm acetic acid, and 2 mm EDTA. Nucleic acids were stained with ethidium bromide. Lane 1, IMAC unbound fraction; lane 2, IMAC wash step; lane 3, IMAC-bound fraction; lane 4, heparin unbound fraction; lane 5, HiTrap Q-bound fraction; lanes 6–8, heparin peak fractions. The positions of size markers (kb) are indicated. C, the purity of untagged p72 protein (∼3.3 μg) after size exclusion chromatography was assessed by 4–20% SDS-PAGE and by staining with Coomassie Brilliant Blue (lane 1) and by immunoblot analysis with polyclonal rabbit antiserum (lane 2), as described under “Experimental Procedures.”

FIGURE 3.

DNA binding affinity of mitochondrial DNA helicase proteins. Changes in the anisotropy of fluorescein-labeled oligonucleotide substrates were measured in response to the stepwise addition of p72 protein, as described under “Experimental Procedures.” Protein concentrations are expressed as hexamers. Baseline anisotropies of single-stranded (TD1-F) and double-stranded (TD1-F·TD2) substrates were 0.072 and 0.039, respectively, reflecting conformational differences in each unbound substrate. Changes in anisotropy of TD1-F (squares) or TD1-F·TD2 (diamonds) due to the addition of wild type p72 protein (closed symbols) or buffer only (open symbols) are shown. Error bars are standard deviations of triplicate determinations. Residual anisotropy following the addition of NaCl to 0.42 m (asterisks) or EDTA to 40 mm (circle) is shown.

FIGURE 4.

DNA helicase activity of mitochondrial DNA helicase proteins. A, the ability of WT p72 to unwind a forked oligonucleotide substrate was determined as described under “Experimental Procedures,” except that reactions contained 3 (lanes 1–4), 6 (lanes 5–8), 12 (lanes 9–12), or 20 nm (lanes 13–16) WT p72 hexamers. Reactions were incubated at 37 °C for 0 (lanes 1, 5, 9, and 13), 5 (lanes 2, 6, 10, and 14), 10 (lanes 3, 7, 11, and 15), or 15 min (lanes 4, 8, 12, and 16) before single-stranded [³²P]TD1 products (P, arrow) and [³²P]TD1·TD3 forked DNA substrates (S, arrow) were resolved by native gel electrophoresis. Substrate denatured by boiling is shown (lane 0). B, products of helicase reactions were quantified as described under “Experimental Procedures” and plotted as a function of time. Reactions contained 3 (circles), 6 (squares), 12 (diamonds), or 20 nm (triangles) WT p72 hexamers. Reactions without enzyme did not exhibit a detectable increase in products after a 15-min incubation. C, DNA helicase activity was determined in standard helicase reactions containing 40 nm forked oligonucleotide substrate and 12 nm of the indicated p72 variant, as described under “Experimental Procedures.” The slopes of independent time course reactions were calculated in duplicate, and helicase velocities were expressed as the fraction of total substrate unwound in 10 min. Error bars are standard deviations of multiple determinations.

FIGURE 5.

ATPase activity of the mitochondrial DNA helicase. A, ATPase activity was assessed as described under “Experimental Procedures.” Reactions (20 μl) contained 191 nm WT p72 monomers (3.82 pmol) and were incubated at 37 °C for 0, 5, 10, 15, or 30 min (lanes 1–5, respectively) prior to thin layer chromatography. Relative mobilities of the ATP substrate and ADP product are indicated. B, ATPase reactions were assembled as in A and included no supplement (circles), 0.2 μg of double-stranded replicative form M13mp18 DNA (squares), 0.2 μg of calf thymus DNA (diamonds), or 0.2 μg of single-stranded viral form M13mp18 DNA (triangles). The time course of each ATPase reaction was quantified as described under “Experimental Procedures.” C, ATPase reactions were supplemented with calf thymus DNA and incubated 30 min with the indicated concentrations of p72 monomers. ATP hydrolysis by WT p72 with (square) and without (circle) a C-terminal His₆ affinity tag was quantified as described under “Experimental Procedures.” D, standard ATPase reactions containing the indicated concentrations of ATP and 245 nm WT p72 monomers (lacking an affinity tag) were incubated for 25 min. Reaction rates are expressed as turnover number for p72 monomers, and data were fit to the Michaelis-Menten steady state rate equation. E, duplicate samples of WT p72 protein were preincubated at 42 °C in the absence of DNA for the indicated times, and ATPase activity was determined in standard reactions incubated for 25 min. Reaction rates are expressed as turnover number for p72 monomers, and data were fit to an equation for exponential decay. Error bars in D and E are standard deviations of at least three determinations.