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


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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Available from: Sergei M Mirkin
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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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    ABSTRACT: Telomeric repeats located within chromosomes are called interstitial telomeric sequences (ITSs). They are polymorphic in length and are likely hotspots for initiation of chromosomal rearrangements that have been linked to human disease. Using our S. cerevisiae system to study repeat-mediated genome instability, we have previously shown that yeast telomeric (Ytel) repeats induce various gross chromosomal rearrangements (GCR) when their G-rich strands serve as the lagging strand template for replication (G orientation). Here, we show that interstitial Ytel repeats in the opposite C orientation prefer to expand rather than cause GCR. A tract of eight Ytel repeats expands at a rate of 4 × 10(-4) per replication, ranking them among the most expansion-prone DNA microsatellites. A candidate-based genetic analysis implicates both post-replication repair and homologous recombination pathways in the expansion process. We propose a model for Ytel repeat expansions and discuss its applications for genome instability and alternative telomere lengthening (ALT).
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    • "Thus, when interpreting mechanisms of CNV formation, it is important to consider the DNA at breakpoints as well as the resulting chromosome rearrangement. Functional analysis of individual DNA motifs will delineate the sequences responsible for gross chromosomal rearrangement [49]–[51]. In addition, motif mining of even larger CNV breakpoint datasets from diverse CNV classes will tell us more about the factors required for CNV formation. "
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    ABSTRACT: Chromosome breakage in germline and somatic genomes gives rise to copy number variation (CNV) responsible for genomic disorders and tumorigenesis. DNA sequence is known to play an important role in breakage at chromosome fragile sites; however, the sequences susceptible to double-strand breaks (DSBs) underlying CNV formation are largely unknown. Here we analyze 140 germline CNV breakpoints from 116 individuals to identify DNA sequences enriched at breakpoint loci compared to 2800 simulated control regions. We find that, overall, CNV breakpoints are enriched in tandem repeats and sequences predicted to form G-quadruplexes. G-rich repeats are overrepresented at terminal deletion breakpoints, which may be important for the addition of a new telomere. Interstitial deletions and duplication breakpoints are enriched in Alu repeats that in some cases mediate non-allelic homologous recombination (NAHR) between the two sides of the rearrangement. CNV breakpoints are enriched in certain classes of repeats that may play a role in DNA secondary structure, DSB susceptibility and/or DNA replication errors.
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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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