Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability, Nucleotide Hydrolysis, and Helicase Activity

Matthew J Longley,Margaret M Humble,Farida S Sharief,William C Copeland

doi:10.1074/jbc.m110.151795

Matthew J Longley, Margaret M Humble + Show 2 more

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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.

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Chronic disruption of mitochondrial function can result in a broad array of neuromuscular degenerative disorders known as mitochondrial diseases, including progressive external ophthalmoplegia (PEO),2 Alpers syndrome, parkinsonism, and several complex ataxia neuropathy syndromes [1,2,3]
Numerous reports have identified 23 additional missense mutations in C10orf2 associated with heritable mitochondrial diseases such as adPEO, hepatocerebral mitochondrial DNA (mtDNA) depletion syndrome (MDS), and infantile-onset spinocerebellar ataxia (IOSCA) [7, 8]
To begin to determine how biochemical defects in the mitochondrial DNA helicase may be contributing to diseases of mtDNA maintenance, we developed a scheme to overproduce wild type (WT) and mutant variants of the mitochondrial DNA helicase in E. coli and to purify the recombinant enzymes to near homogeneity

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Chronic disruption of mitochondrial function can result in a broad array of neuromuscular degenerative disorders known as mitochondrial diseases, including progressive external ophthalmoplegia (PEO), Alpers syndrome, parkinsonism, and several complex ataxia neuropathy syndromes [1,2,3]. In 2001, autosomal dominant PEO with mtDNA deletions was shown to co-segregate with 11 different missense mutations in the C10orf gene [5], which encoded a protein containing predicted amino acid sequences homologous to portions of the bacteriophage T7 gene product 4 and other superfamily 4 DNA helicases [5, 6]. Overexpression of catalytic mutants and dominant disease variants of the mtDNA helicase in cultured human or Schneider cells results in stalled mtDNA replication or depletion of mtDNA [11,12,13], which emulates the disease state. Overexpression of two adPEO variants in a mouse transgenic model recapitulated many of the symptoms observed in human PEO, including accumulation of multiple mtDNA deletions, progressive respiratory dysfunction, and cytochrome c oxidase deficiency [14, 15]. Variability in protocols for protein purification and differences in DNA substrates and assay conditions make it difficult to reconcile these discrepancies

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