GAA TTC STRUCTURES--FUNCTIONS AND FRIEDREICHS ATAXIA

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Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by an intronic GAA.TTC triplet repeat expansion. FRDA is the most common hereditary ataxia. The FRDA gene is spread over 40 kb and contains five exons which encode a 210 amino acid protein named frataxin. Normal individuals contain 7-22 GAA.TTC units whereas FRDA patients have enlarge and polymorphic alleles with 100-1700 units. The expansion of the GAA.TTC repeat in the first intron of the frataxin gene results in reduced levels of frataxin mRNA and protein. Shorter lengths of GAA.TTC repeats adopt intra- and inter-molecular triplexes and long repeats (>60 units) form one of several possible unusual conformations. Our recent discovery that 150 and 270 repeats of GAA.TTC adopt a dramatically new form of "bent" DNA heightens the interest in the role of DNA conformation in the molecular etiology of this disease. The goal is to elucidate the nucleic acid molecular mechanisms responsible for FRDA. Aim 1 is to study the conformations of different lengths of GAA.TTC from normal individuals to the enlarged alleles. Characterization will be by hydrodynamic properties, chemical and enzymatic probe analysis, EM, circularization kinetics and helical repeat studies. Also, the conformations formed between DNA GAA.TTC and intronic rGAA will be investigated. Aim 2 is to study the transcription properties of the FRDA TRS structures by nuclear runoff experiments from patient materials, determinations of the transcription elongation rate in vitro, and studies on the RNA as well as mRNA.DNA structures. Also, the fidelity of transcription will be monitored along with the effects of transcription on repeat stability. Our new data revealed the inhibitory effects of expanded intronic genetic instabilities of the FRDA triplet repeat sequences (TRS) in vivo and vitro. In summary, we shall study the molecular etiology of FRDA by elucidating the DNA conformations of different lengths of GAA.TTC and their effect on transcription and on genetic instabilities (replication and recombination).