Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene. We created an experimental system to analyze expansions of GAA•TTC repeats in cultured human cells. It employs a shuttle plasmid that can replicate in human cells or be stably maintained in S. cerevisiae , which also contains a selectable cassette allowing us to detect repeat expansions that accumulated in human cells upon plasmid transformation into yeast. We observed massive expansions of GAA•TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAA•TTC repeats stall replication fork progression, while the frequency of repeat expansions appears to depend on proteins implicated in replication fork stalling, reversal, and restart. Locked nucleic acid (LNA)-DNA mixmer oligonucleotides, which interfere with triplex formation by GAA•TTC repeats, prevented the expansion of these repeats in human cells. We hypothesize that triplex formation by GAA•TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart.
.png)
