Sticky DNA, a Self-associated Complex Formed at Long GAA·TTC Repeats in Intron 1 of the Frataxin Gene, Inhibits Transcription

Abstract

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by the expansion of GAA⅐TTC repeats in the first intron of the frataxin (X25) gene. FRDA patients carrying two expanded GAA⅐TTC repeats show very low levels of mature frataxin mRNA and protein. A novel type of unusual DNA structure, sticky DNA, was previously found in the expanded GAA⅐TTC repeats from FRDA patients. To evaluate the effect of sticky DNA on transcription, in vitro transcription studies of (GAA⅐TTC) n repeats (where n ‫؍‬ 9 -150) were carried out using T7 or SP6 RNA polymerase. When a gel-isolated sticky DNA template was transcribed, the amount of full-length RNA synthesized was significantly reduced compared with the transcription of the linear template. Surprisingly, transcriptional inhibition was observed not only for the sticky DNA template but also another DNA molecule used as an internal control in an orientation-independent manner. The molecular mechanism of transcriptional inhibition by sticky DNA was a sequestration of the RNA polymerases by direct binding to the complex DNA structure. Moreover, plasmids containing the (GAAGGA⅐TCCTTC) 65 repeat, which does not form sticky DNA, did not inhibit in vitro transcription, as expected. These results suggest that the role of sticky DNA in FRDA may be the sequestration of transcription factors.Friedreich's ataxia (FRDA), 1 an autosomal recessive neurodegenerative disease, is the most common inherited ataxia and is characterized by progressive gait and limb ataxia, dysarthria, lower limb areflexia, diminished vibration sense, muscle weakness of the legs, and extensor planter response (1-4). The gene responsible for FRDA was mapped to chromosome 9q13-q21.1 by linkage studies (5, 6). The FRDA gene, X25, contains seven exons (1-6) and encodes a 210-amino acid protein, frataxin (7). The majority (about 98%) of FRDA patients have an expanded GAA⅐TTC repeat in the first intron of the frataxin gene. Normal alleles have 6 -34 repeats of uninterrupted GAA⅐TTC triplets, whereas FRDA-associated alleles have 66 -1700 or more GAA⅐TTC repeats (7-10). The recessive inheritance and the sequence and the location of the repeat make FRDA unusual among all known triplet diseases (11). A unique molecular mechanism for the etiology of FRDA has been proposed (7,11).An inverse correlation between the length of the GAA⅐TTC repeats and the age of onset and the severity of the disease has been demonstrated (8, 12, 13). FRDA patients carrying expanded GAA⅐TTC repeats in both alleles have reduced levels of the frataxin protein, and there is an inverse correlation between the size of GAA⅐TTC repeat and the amount of frataxin protein (14). The reduction in the amount of the frataxin protein in FRDA patients is due to the reduction in the amount of the frataxin mRNA (7,15,16). Recent work revealed that the amount of the X25 mRNA is inversely related to the length of the GAA⅐TTC repeat in vitro (17) and in vivo (18). The formation of three-stranded DNA structures has been proposed to be involved i...

Structure-dependent Recombination Hot Spot Activity of GAA·TTC Sequences from Intron 1 of the Friedreich's Ataxia Gene

“…Hence, two opposing effects of novobiocin were found: an inhibition of recombination between the short GAA⅐TTC tracts and a stimulation of this process for longer repeats. In the control experiments performed with CTG⅐CAG repeats of different lengths (17,67, and 98 triplets), the in vivo relaxation of the plasmids in the presence of novobiocin led to a 2-4-fold decrease in the frequency of intramolecular recombination (data not shown). Therefore, the behavior found for the GAA⅐TTC containing plasmids (Fig.…”

“…2B). To identify the RB, the cleaved DNA samples were incubated for 10 min at 80°C in 25 mM EDTA (pH 8.0) as described earlier (15)(16)(17)(18)(19). Under these conditions, RB is completely converted to the linear restriction fragments (Fig.…”

“…The structural properties of the GAA⅐TTC repeats were related to their pathogenic potential. The role of intramolecular triplexes (11, 12), DNA/RNA hybrid triplexes (11,13,14), and a sticky DNA structure (15)(16)(17)(18)(19)(20) influenced the instability of the GAA⅐TTC tracts as well as the expression of the FRDA gene.Recombination, together with replication and repair, is one of the major processes responsible for the instability of microsatellite sequences (10). In fact, expansions, deletions, and exchange of point mutations between two CTG⅐CAG tracts were found in yeast (21-24), mammalian cells (25, 26), and in Escherichia coli using an intramolecular genetic assay as well as two-plasmid recombination systems (27)(28)(29)(30)(31).…”

“…The structural properties of the GAA⅐TTC repeats were related to their pathogenic potential. The role of intramolecular triplexes (11, 12), DNA/RNA hybrid triplexes (11,13,14), and a sticky DNA structure (15)(16)(17)(18)(19)(20) influenced the instability of the GAA⅐TTC tracts as well as the expression of the FRDA gene.…”

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

“…Also, a reduction in the amount of the frataxin protein, a mitochondrial protein involved in iron metabolism, is because of a diminution in the abundance of the frataxin mRNA (1). This inhibition of transcription is caused by the capacity of long GAA⅐TTC repeats to form triplexes (three-stranded DNA structures) that are known to effect this inhibition (5,(7)(8)(9)(10)(11)(12). FRDA is the only triplet repeat disease that has a recessive inheritance pattern and is caused by the expansion of a GAA⅐TTC repeat (6).…”