Article

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

Department of Biology, Tufts University, Medford, MA 02155, USA.
Molecular cell (Impact Factor: 14.02). 08/2009; 35(1):82-92. DOI: 10.1016/j.molcel.2009.06.017
Source: PubMed

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 knockouts of postreplication DNA repair proteins, Rad6 and Rad5, and the DNA helicase Sgs1. We propose a model for large-scale repeat expansions based on template switching during replication fork progression through repetitive DNA.

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    • "In Ytel's case, however, the presence of just 15 copies of the repeat in the C orientation resulted in the complete inactivation of the URA3 gene (Figure 1B). This result was unexpected, since the total length of the intron in this case corresponded to only 475 bp, which was considerably below the splicing threshold (Shishkin et al., 2009; Yu and Gabriel, 1999). Also, yeast clones having 15 Ytel repeats within the intron in the opposite G orientation remained Ura + (Aksenova et al., 2013). "
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    • "These RTEL1 findings—inhibition of expansions , hairpin unwinding, and function with the Rad5 ortholog HLTF and Rad18—all closely resemble yeast Srs2, indicating the strong functional conservation between the two enzymes with respect to (CTG,CAG) repeat expansions, despite their lack of protein sequence homology. We note that mutations in yeast SRS2, RAD5, or RAD18 have different mutagenic effects depending on the type of microsatellite (Cherng et al., 2011; Daee et al., 2007; Shishkin et al., 2009), perhaps due to the different DNA structures they can adopt (Mirkin, 2007). We found expression of RTEL1 in yeast srs2 mutants efficiently suppressed MMS sensitivity and CAG-repeat-dependent chromosomal fragility and expansions, whereas expression of Fbh1 was substantially poorer in reversing these phenotypes (Figure 3). "
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