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.


Available from: Sergei M Mirkin
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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.
    PLoS ONE 07/2014; 9(7):e101607. DOI:10.1371/journal.pone.0101607 · 3.23 Impact Factor
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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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    ABSTRACT: Human RTEL1 is an essential, multifunctional helicase that maintains telomeres, regulates homologous recombination, and helps prevent bone marrow failure. Here, we show that RTEL1 also blocks trinucleotide repeat expansions, the causal mutation for 17 neurological diseases. Increased expansion frequencies of (CTG⋅CAG) repeats occurred in human cells following knockdown of RTEL1, but not the alternative helicase Fbh1, and purified RTEL1 efficiently unwound triplet repeat hairpins in vitro. The expansion-blocking activity of RTEL1 also required Rad18 and HLTF, homologs of yeast Rad18 and Rad5. These findings are reminiscent of budding yeast Srs2, which inhibits expansions, unwinds hairpins, and prevents triplet-repeat-induced chromosome fragility. Accordingly, we found expansions and fragility were suppressed in yeast srs2 mutants expressing RTEL1, but not Fbh1. We propose that RTEL1 serves as a human analog of Srs2 to inhibit (CTG⋅CAG) repeat expansions and fragility, likely by unwinding problematic hairpins.
    Cell Reports 02/2014; 6(5). DOI:10.1016/j.celrep.2014.01.034 · 8.36 Impact Factor
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    • "Furthermore, there is no substantial evidence that the expanded (GAA)n repeats affect the RNA splicing of the FXN gene. Minigene constructs transfected into mammalian cells (Baralle et al. 2008) as well as a yeast gene fused with expanded (GAA)n repeats (Shishkin et al. 2009) exhibited splicing differences in the case of expanded repeats; however, these findings were not reproduced in the native context of an intact FXN locus (Bidichandani et al. 1998; Punga and Buhler 2010). One way of explaining reduced FXN transcription in FRDA could be the physical-blockage effects of (GAA)n repeats on the RNAPII transcription machinery. "
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    ABSTRACT: This is an exciting time in the study of Friedreich's ataxia. Over the last 10 years much progress has been made in uncovering the mechanisms, whereby the Frataxin gene is silenced by (GAA)n repeat expansions and several of the findings are now ripe for testing in the clinic. The discovery that the Frataxin gene is heterochromatinised and that this can be antagonised in vivo has led to the tantalizing possibility that the disease might be amenable to a more radical therapeutic approach involving epigenetic modifiers. Here, we set out to review progress in the understanding of the fundamental mechanisms whereby genes are regulated at this level and how these findings have been applied to achieve a deeper understanding of the dysregulation that occurs as the primary genetic lesion in Friedreich's ataxia.
    Journal of Neurochemistry 08/2013; 126(s1). DOI:10.1111/jnc.12254 · 4.28 Impact Factor
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