We propose that a nick upstream of the replication fork transforms into a double-strand break during replication and expansion happens during the misalignment of the GAA repeats during the repair of this break via homologous recombination. 20/n

Turns out, homologous recombination is mostly required for all the expansions where the replication fork bumps into the nick first. This means that the position relative to replication is what determines the mechanism of expansion and not the composition of the strand! 19/n

In our original cassette, the replication machine first bumps in the 5’-GAA nick and then into the repeat, and in the inverted cassette, the replication fork first bumps into the repeat and then into the 5’-GAA nick. The situation is mirrored for the 5’-TTC nick. 18/n

Even among the two 5’-nicks, the mechanisms by which they cause expansions are different: the 5’-nick near the GAA repeat strand (and much less for the TTC strand) promotes expansions that depend on the Rad52 protein and thus happen via homologous recombination. 16/n

We found that not all nicks are created equal! Turns out, the nicks at the 5’- end of either strand lead to much higher GAA repeat expansion than those on the 3’-ends. 15/n

Even more exciting, when we induce a nick near shorter tracts of GAA repeats, such as 40xGAA or 33xGAA repeats, we still detect a bunch of expansion events. This is the first time someone ever detected an expansion of a pre-mutational GAA repeat allele in a model system! 13/n

We found that expressing the nickase HUGELY increases the rate of long 100xGAA repeat tract expansion, by up to ∼340 fold (when I said hugely I meant it!). This makes the rate of expansion comparable with those observed in human pedigrees. 12/n

To overcome this problem, the @semirkin.bsky.social lab previously developed an extremely sensitive system in yeast that allows to ‘catch’ GAA repeat expansion happening with a probability of up to 10^-7 per cell per generation. 9/n

The short GAA repeat alleles remain stable during intergenerational transitions. In contrast, the long, pathogenic alleles change their length with almost 100% probability and often become longer in children compared to their parents. 6/n

While most people have less than 33 GAA repeats in this location, Friedreich’s ataxia patients have more than 70 repeats, and occasionally more than 1000 GAA repeats! Such a large expansion turns off the FXN gene and leads to disability and ultimately death of a patient. 5/n