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

Article (PDF Available)inMolecular cell 35(1):82-92 · August 2009with75 Reads
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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    • "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). "
    Full-text · Article · Aug 2016
    • "Moreover in FRDA cells the activation of dormant origins acts a safeguarding mechanism assuring the replication of the FXN gene. The consequence is that the number of forks running in the opposite direction through the GAA-repeat is increased (S7 and S8 Figs) and this sequence is replicated preferentially from the leading strand template, an orientation not frequently involved in fork pausing according to model systems [11,55]. Thus, in spite of the weak evidence available from this study, the detection of fork arrest events in proximity of the short GAA-repeat in GM15851 control cell line must be taken into further consideration as a possible impact of the short non-pathological GAA-repeat on fork progression. "
    [Show abstract] [Hide abstract] ABSTRACT: Author The expansion of trinucleotide repeats (TNR) is associated with a large number of human neurodegenerative and neuromuscular diseases, among which the most known are Friedreich's ataxia (GAA/TTC), Huntington's disease (CAG/CTG), and myotonic dystrophy (CTG/CAG). TNR are among the most unstable DNA regions in the genome, and an important step in their expansion is the attainment of a threshold-length. This process occurs during paternal or maternal gametogenesis, leading to an earlier onset of the disease in the next generation. The severity of the disease is strictly related to the TNR length. The repeat instability results from non-B DNA secondary structures formed during DNA replication, repair, recombination and gene transcription. However, the pathways leading to expansion remain poorly understood. Here, we describe the effects of the GAA-expansion on the DNA replication of Frataxin gene. By analyzing the replication profile of mutated and normal genotypes, we have found that the replication of the expanded gene is slowed or delayed in comparison with the non-expanded condition. Interestingly, this observation accords to a global modification of the replication profile affecting the usage of both replication forks and origins, which can be referred as the functional units of any replication program.
    Full-text · Article · Jul 2016
    • "The DNA Replication Program Is Altered at the Endogenous FXN Locus in FRDA iPSCs In vitro models predict that repeat expansion is caused by DNA polymerase slippage at stalled replication forks (Mirkin, 2007). Using a plasmid-based model system, it has been shown that the replication fork stalls at expanded GAA repeats (Chandok et al., 2012; Krasilnikova and Mirkin, 2004 ) and that altered replication fork progression results in repeat instability (Cleary et al., 2002; Pelletier et al., 2003; Rindler et al., 2006; Shishkin et al., 2009 ). It is still unknown, however, how the replication fork proceeds through the endogenous FXN locus in the native chromatin environment and whether the replication machinery stalls at the GAA repeats in FRDA cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Friedreich’s ataxia (FRDA) is caused by the expansion of GAA repeats located in the Frataxin (FXN) gene. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism leading to repeat expansion and decreased FXN transcription remains unclear. Using single-molecule analysis of replicated DNA, we detected that expanded GAA repeats present a substantial obstacle for the replication machinery at the FXN locus in FRDA cells. Furthermore, aberrant origin activation and lack of a proper stress response to rescue the stalled forks in FRDA cells cause an increase in 3′-5′ progressing forks, which could enhance repeat expansion and hinder FXN transcription by head-on collision with RNA polymerases. Treatment of FRDA cells with GAA-specific polyamides rescues DNA replication fork stalling and alleviates expansion of the GAA repeats, implicating DNA triplexes as a replication impediment and suggesting that fork stalling might be a therapeutic target for FRDA.
    Full-text · Article · Jul 2016
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