Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease. Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA. These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro. Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1. 1 MDa (megadaltons) and a diameter of 13+/-2 nm. Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form. In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.

We show that GAA instability in Friedreich's Ataxia is a DNA-directed mutation caused by improper DNA structure at the repeat region. Unlike CAG or CGG repeats, which form hairpins, GAA repeats form a YRY triple helix containing non-Watson-Crick pairs. As with hairpins, triplex mediates intergenerational instability in 96% of transmissions. In families with Friedreich's Ataxia, the only recessive trinucleotide disease, GAA instability is not a function of the number of long alleles, ruling out homologous recombination or gene conversion as a major mechanism. The similarity of mutation pattern among triple repeat-related diseases indicates that all trinucleotide instability occurs by a common, intraallelic mechanism that depends on DNA structure. Secondary structure mediates instability by creating strong polymerase pause sites at or within the repeats, facilitating slippage or sister chromatid exchange.

We have directly compared intergenerational stability of intermediate alleles (IAs) derived from new mutation families (IANM) for Huntington disease (HD) with IAs in the general population (IAGP) which occur in approximately 1 in 50 persons. Analysis of meiotic events in blood and sperm reveals that IANM are significantly more unstable than IAGP despite similar size. However, for both IANM and IAGP CAG changes were small and risks for inheriting an expansion into the HD affected range were low. Sequence analysis reveals that the CAG tract is generally interrupted by a penultimate CAA in IAGP, IANM and alleles in the affected range. In one new mutation family, however, two A-->G mutations result in a pure CAG tract which is associated with very marked instability. These mutations alter the predicted DNA hairpin structure with a predicted increase in the likelihood of large expansion, supporting the model that hairpin loop formation plays an important role in trinucleotide instability.

Frataxin is a conserved mitochondrial protein required for iron homeostasis. We showed previously that in the presence of ferrous iron recombinant yeast frataxin (mYfh1p) assembles into a regular multimer of approximately 1.1 MDa storing approximately 3000 iron atoms. Here, we further demonstrate that mYfh1p and iron form a stable hydrophilic complex that can be detected by either protein or iron staining on nondenaturing polyacrylamide gels, and by either interference or absorbance measurements at sedimentation equilibrium. The molecular mass of this complex has been refined to 840 kDa corresponding to 48 protein subunits and 2400 iron atoms. Solution density measurements have determined a partial specific volume of 0.58 cm(3)/g, consistent with the amino acid composition of mYfh1p and the presence of 50 Fe-O equivalents per subunit. By dynamic light scattering, we show that the complex has a radius of approximately 11 nm and assembles within 2 min at 30 degrees C when ferrous iron, not ferric iron or other divalent cations, is added to mYfh1p monomer at pH between 6 and 8. Iron-rich granules with diameter of 2-4 nm are detected in the complex by scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy. These findings support the hypothesis that frataxin is an iron storage protein, which could explain the mitochondrial iron accumulation and oxidative damage associated with frataxin defects in yeast, mouse, and humans.

We show that repeating units from all reported disease genes are capable of forming hairpins of common structure and threshold stability. The threshold stability is roughly -50 kcal per hairpin and is influenced by the flanking sequence of the gene. Hairpin stability has two components, sequence and length; only DNA of select sequences and the correct length can form hairpins of threshold energy. There is a correlation among the ability to form hairpins of threshold stability, the sequence selectivity of expansion, and the length dependence of expansion. Additionally, hairpin formation provides a potential structural basis for the constancy of the CCG region of the Huntington's disease gene in individuals and explains the stabilizing effects of AGG interruptions in FMR1 alleles.