Large-Scale Expansions of Friedreich's Ataxia GAA Repeats in Yeast

Abstract

Large-scale expansions of DNA repeats are implicated in numerous hereditary disorders in humans. We describe a yeast experimental system to analyze large-scale expansions of triplet GAA repeats, responsible for the human disease Friedreich’s ataxia. When GAA repeats were placed into an intron of the chimeric URA3 gene, their expansions caused gene inactivation, which was detected on the selective media. We found that the rates of expansions of GAA repeats increased exponentially with their lengths. These rates were only mildly dependent on the repeat’s orientation within the replicon, whereas the repeat-mediated replication fork stalling was exquisitely orientation-dependent. Expansion rates were significantly elevated upon inactivation of the replication fork stabilizers, Tof1 and Csm3, but decreased in the mutants of postreplication DNA repair proteins, Rad6 and Rad5, and the DNA helicase Sgs1. We propose a model for large-scale repeat expansions based on the template switching during the replication fork progression through repetitive DNA.

Genome rearrangements caused by interstitial telomeric sequences in yeast

“…Similar induction of mutagenesis was observed for (GAA) n and inverted repeats [repeat-induced mutagenesis (RIM)] (11,(13)(14)(15). All these repeats stall the replication fork progression (24)(25)(26), and the (TGTGTGGG) 15 run is a particularly potent replication block.…”

“…The reporter gene used to detect changes within the interstitial Ytel sequences is closely related to the one used previously to detect expansions of the (GAA) n repeats (11), which were inserted into an intron of the artificially split URA3 gene (construction details are in SI Methods, Tables S1 and S2). Because long (>1-kb) introns are inefficiently removed from pre-mRNA in yeast (12), large-scale expansions of the (GAA) n tracts produce Ura − clones that might be selected on the medium containing 5-fluoroorotic acid (5-FOA) (11).…”

“…Because long (>1-kb) introns are inefficiently removed from pre-mRNA in yeast (12), large-scale expansions of the (GAA) n tracts produce Ura − clones that might be selected on the medium containing 5-fluoroorotic acid (5-FOA) (11). 5-FOA-resistant clones also resulted from large deletions around those repeats (11,13), as well as from the repeatinduced mutagenesis in the body of the URA3 gene (13)(14)(15).…”

“…Although the natural yeast telomeres do not have identical copies of a small repeat, we constructed reporter genes that had either 8 or 15 copies of the perfect octameric repeat (TGTGTGGG), which is complementary to the telomeric RNA template within telomerase (16). The (TGTGTGGG) n sequence was in the nontranscribed strand of URA3 placing it onto the lagging strand template for replication forks emanating from ARS306 responsible for replication of our reporter (11). Yeast strains with…”

“…Second, ITSs stimulate mutations in flanking sequences. Several studies recently showed that double-strand DNA breaks (DSBs) in long (GAA) n tracts (11,(13)(14)(15) or inverted repeats (14) elevate rates of mutations in closely linked genes. It is likely that ITSs operate by related mechanisms.…”