Sticky DNA, a Long GAA·GAA·TTC Triplex That Is Formed Intramolecularly, in the Sequence of Intron 1 of the Frataxin Gene

Abstract

Friedreich's ataxia is caused by the massive expansion of GAA⅐TTC repeats in intron 1 of the frataxin (X25) gene. Our prior investigations showed that long GAA⅐TTC repeats formed very stable triplex structures which caused two repeat tracts to adhere to each other (sticky DNA). This process was dependent on negative supercoiling and the presence of divalent metal ions. Herein, we have investigated the formation of sticky DNA from plasmid monomers and dimers; sticky DNA is formed only when two tracts of sufficiently long (GAA⅐TTC) n (n ‫؍‬ 59 -270) are present in a single plasmid DNA and are in the direct repeat orientation. If the inserts are in the indirect (inverted) repeat orientation, no sticky DNA was observed. Furthermore, kinetic studies support the intramolecular nature of sticky DNA formation. Electron microscopy investigations also provide strong data for sticky DNA as a single long triplex. Hence, these results give new insights into our understanding of the capacity of sticky DNA to inhibit transcription and thereby reduce the level of frataxin protein as related to the etiology of Friedreich's ataxia.Friedreich's ataxia (FRDA) 1 is the most common hereditary ataxia. The gene for this autosomal recessive neurodegenerative disease has been mapped to chromosome 9q13-q21.1 by linkage studies (1, 2). The clinical features and molecular biology FRDA have been reviewed (3-10). The FRDA gene, X25, contains seven exons and encodes a 210-amino acid protein called frataxin (8). The vast majority of FRDA patients have an expanded GAA⅐TTC repeat in the first intron of the frataxin gene. Normal alleles have 6 -34 repeats of GAA⅐TTC repeats, but FRDA-associated alleles have 66 -1,700 or more GAA⅐TTC repeats (3,8,10). FRDA is the only triplet repeat disease that has a recessive inheritance pattern and is caused by the expansion of a GAA⅐TTC repeat (9).The age of onset and the severity of the disease are correlated with the length of the GAA⅐TTC repeats (8,11,12). Patients carrying expanded GAA⅐TTC repeats in both alleles have reduced levels of frataxin, and an inverse correlation exists between the size of the GAA⅐TTC repeat and amount of the frataxin protein (13,14). This reduction in the amount of the frataxin protein is caused by a diminution in the amount of the frataxin mRNA (8,15,16). The amount of the X25 mRNA is inversely related to the length of the GAA⅐TTC repeat both in vitro (17) and in vivo (18). Virtually all workers in the field have proposed that the long GAA⅐TTC repeats form triplexes (three stranded DNA structures) that are involved in the transcriptional inhibition (16 -22). In fact, recent investigations (22) directly demonstrated that sticky DNA (a complex triplex formed at GAA⅐TTC repeats; described below) inhibits transcription in vitro, and in vivo investigations (18, 23) are consistent with these results.Substantial prior investigations (19, 24 -29) showed that shorter GAA⅐TTC inserts (16 -58 repeats) adopt triplex structures. DNA triplexes are formed at polypurine⅐polypyrimidine (R⅐Y) t...

Sticky DNA Formation in Vivo Alters the Plasmid Dimer/Monomer Ratio

“…RB is converted completely to the linear monomeric form after incubation of the DNA for 10 min at 80°C in 50 mM EDTA as described earlier (8,10,13,14). Thus, the heat sensitivity of RB in the absence of divalent metal ions allows us to distinguish between RB and other possible structures.…”

“…The requirement for two blocks of long GAA⅐TTC repeats can be fulfilled by either having two tracts in one recombinant plasmid or by biological dimers of the plasmids, each parent monomer containing a single repeat tract. The intermolecular formation of sticky DNA from two independent plasmid molecules, each harboring a single long GAA⅐TTC tract, has never been observed (13,14). The novel feature of sticky DNA is the association into a triplex of two long R⅐Y tracts from distant regions of a DNA molecule; hence, upon its linearization, an X-shaped structure (8, 13) is formed with the crossover point at the repeat sequences.…”

“…The intermolecular formation of sticky DNA from two independent plasmid molecules, each harboring a single long GAA⅐TTC tract, has never been observed (13,14). The novel feature of sticky DNA is the association into a triplex of two long R⅐Y tracts from distant regions of a DNA molecule; hence, upon its linearization, an X-shaped structure (8,13) is formed with the crossover point at the repeat sequences. After the sticky DNA conformation has been formed, it is remarkably stable and requires the removal of divalent metal ions, with EDTA, as well as heat (70°C) to revert the conformation to the duplex.…”

“…Hoogsteen base-pairing stabilizes the GAA⅐GAA interactions, whereas the GAA⅐TTC pairing is Watson-Crick. Two long tracts of (GAA⅐TTC) n (where n ϭ 59 -270) in the direct repeat orientation in a single DNA molecule are required for the formation of sticky DNA (13,14); if the tracts are in the indirect repeat orientation, sticky DNA cannot be formed (13,14). The requirement for two blocks of long GAA⅐TTC repeats can be fulfilled by either having two tracts in one recombinant plasmid or by biological dimers of the plasmids, each parent monomer containing a single repeat tract.…”

Non-B DNA Conformations, Genomic Rearrangements, and Human Disease

“…This tract, which occurs in intron 1, is well known to adopt the triplex conformation (Fig. 1) as well as the more complex sticky DNA structure between two long GAA⅐TTC tracts at distant locations of the same molecule (35,36). All workers in this field agree (30) that the triplex and/or sticky DNA conformation adopted by this repeating sequence is involved in the inhibition of transcription of the Friedreich's ataxia gene to give a reduction in the amount of the frataxin protein.…”