Replication-independent instability of Friedreich’s ataxia GAA repeats during chronological aging

Nearly 50 hereditary diseases result from the inheritance of abnormally long repetitive DNA microsatellites. While it was originally believed that the size of inherited repeats is the key factor in disease development, it has become clear that somatic instability of these repeats throughout an individual’s lifetime strongly contributes to disease onset and progression. Importantly, somatic instability is commonly observed in terminally differentiated, postmitotic cells, such as neurons. To unravel the mechanisms of repeat instability in nondividing cells, we created an experimental system to analyze the mutability of Friedreich’s ataxia (GAA)n repeats during chronological aging of quiescent Saccharomyces cerevisiae. Unexpectedly, we found that the predominant repeat-mediated mutation in nondividing cells is large-scale deletions encompassing parts, or the entirety, of the repeat and adjacent regions. These deletions are caused by breakage at the repeat mediated by mismatch repair (MMR) complexes MutSβ and MutLα and DNA endonuclease Rad1, followed by end-resection by Exo1 and repair of the resulting double-strand breaks (DSBs) via nonhomologous end joining. We also observed repeat-mediated gene conversions as a result of DSB repair via ectopic homologous recombination during chronological aging. Repeat expansions accrue during chronological aging as well—particularly in the absence of MMR-induced DSBs. These expansions depend on the processivity of DNA polymerase δ while being counteracted by Exo1 and MutSβ, implicating nick repair. Altogether, these findings show that the mechanisms and types of (GAA)n repeat instability differ dramatically between dividing and nondividing cells, suggesting that distinct repeat-mediated mutations in terminally differentiated somatic cells might influence Friedreich’s ataxia pathogenesis.

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Friedreich's Ataxia Induced Pluripotent Stem Cells Model Intergenerational GAA⋅TTC Triplet Repeat Instability

Cell Stem Cell ◽

10.1016/j.stem.2010.09.014 ◽

2010 ◽

Vol 7 (5) ◽

pp. 631-637 ◽

Cited By ~ 148

Author(s):

Sherman Ku ◽

Elisabetta Soragni ◽

Erica Campau ◽

Elizabeth A. Thomas ◽

Gulsah Altun ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Friedreich’S Ataxia ◽

Friedreich's Ataxia ◽

Triplet Repeat ◽

Repeat Instability ◽

Induced Pluripotent

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Large-scale contractions of Friedreich’s ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913416117 ◽

2020 ◽

Vol 117 (3) ◽

pp. 1628-1637 ◽

Cited By ~ 1

Author(s):

Alexandra N. Khristich ◽

Jillian F. Armenia ◽

Robert M. Matera ◽

Anna A. Kolchinski ◽

Sergei M. Mirkin

Keyword(s):

Dna Replication ◽

Genetic Analysis ◽

Dna Polymerase ◽

Mirror Symmetry ◽

Large Scale ◽

Catalytic Domain ◽

Friedreich’S Ataxia ◽

Hereditary Disease ◽

Friedreich's Ataxia ◽

Lagging Strand

Friedreich’s ataxia (FRDA) is a human hereditary disease caused by the presence of expanded (GAA)n repeats in the first intron of the FXN gene [V. Campuzano et al., Science 271, 1423–1427 (1996)]. In somatic tissues of FRDA patients, (GAA)n repeat tracts are highly unstable, with contractions more common than expansions [R. Sharma et al., Hum. Mol. Genet. 11, 2175–2187 (2002)]. Here we describe an experimental system to characterize GAA repeat contractions in yeast and to conduct a genetic analysis of this process. We found that large-scale contraction is a one-step process, resulting in a median loss of ∼60 triplet repeats. Our genetic analysis revealed that contractions occur during DNA replication, rather than by various DNA repair pathways. Repeats contract in the course of lagging-strand synthesis: The processivity subunit of DNA polymerase δ, Pol32, and the catalytic domain of Rev1, a translesion polymerase, act together in the same pathway to counteract contractions. Accumulation of single-stranded DNA (ssDNA) in the lagging-strand template greatly increases the probability that (GAA)n repeats contract, which in turn promotes repeat instability in rfa1, rad27, and dna2 mutants. Finally, by comparing contraction rates for homopurine-homopyrimidine repeats differing in their mirror symmetry, we found that contractions depend on a repeat’s triplex-forming ability. We propose that accumulation of ssDNA in the lagging-strand template fosters the formation of a triplex between the nascent and fold-back template strands of the repeat. Occasional jumps of DNA polymerase through this triplex hurdle, result in repeat contractions in the nascent lagging strand.

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Rad9-mediated checkpoint activation is responsible for elevated expansions of GAA repeats in CST-deficient yeast

Genetics ◽

10.1093/genetics/iyab125 ◽

2021 ◽

Author(s):

Ekaterina Spivakovsky-Gonzalez ◽

Erica J Polleys ◽

Chiara Masnovo ◽

Jorge Cebrian ◽

Adrian M Molina-Vargas ◽

...

Keyword(s):

Large Scale ◽

Experimental System ◽

Fold Increase ◽

Telomere Maintenance ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Checkpoint Activation ◽

Repeat Instability ◽

Repeat Expansions ◽

Gaa Repeats

Abstract Large-scale expansion of (GAA)n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich’s ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system (Zhang et al. 2012). The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions. This screen, however, also produced two unexpected hits: members of the CST complex, CDC13 and TEN1 genes, which are required for telomere maintenance. To understand how the CST complex could affect intra-chromosomal GAA repeats, we studied the well-characterized temperature-sensitive cdc13-1 mutation and its effects on GAA repeat instability in yeast. We found that, in-line with the screen results, this mutation leads to ∼10-fold increase in the rate of large-scale expansions of the (GAA)100 repeat at semi-permissive temperature. Unexpectedly, the hyper-expansion phenotype of the cdc13-1 mutant largely depends on activation of the G2/M checkpoint, as deletions of individual genes RAD9, MEC1, RAD53 and EXO1 belonging to this pathway rescued the increased GAA expansions. Further, the hyper-expansion phenotype of the cdc13-1 mutant depended on the subunit of DNA Polymerase δ, Pol32. We hypothesize, therefore, that increased repeat expansions in the cdc13-1 mutant happen during post-replicative repair of nicks or small gaps within repetitive tracts during the G2 phase of the cell cycle upon activation of the G2/M checkpoint.

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

Molecular Cell ◽

10.1016/j.molcel.2009.06.017 ◽

2009 ◽

Vol 35 (1) ◽

pp. 82-92 ◽

Cited By ~ 96

Author(s):

Alexander A. Shishkin ◽

Irina Voineagu ◽

Robert Matera ◽

Nicole Cherng ◽

Brook T. Chernet ◽

...

Keyword(s):

Large Scale ◽

Friedreich’S Ataxia ◽

Friedreich's Ataxia ◽

Gaa Repeats

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An Overview of the Ferroptosis Hallmarks in Friedreich’s Ataxia

Biomolecules ◽

10.3390/biom10111489 ◽

2020 ◽

Vol 10 (11) ◽

pp. 1489

Author(s):

Riccardo Turchi ◽

Raffaella Faraonio ◽

Daniele Lettieri-Barbato ◽

Katia Aquilano

Keyword(s):

Reactive Oxygen Species ◽

Mitochondrial Protein ◽

Disease Onset ◽

Friedreich’S Ataxia ◽

Therapeutic Strategies ◽

Friedreich's Ataxia ◽

Early Mortality ◽

Molecular Characteristics ◽

Oxygen Species ◽

Molecular Alterations

Background: Friedreich’s ataxia (FRDA) is a neurodegenerative disease characterized by early mortality due to hypertrophic cardiomyopathy. FRDA is caused by reduced levels of frataxin (FXN), a mitochondrial protein involved in the synthesis of iron-sulphur clusters, leading to iron accumulation at the mitochondrial level, uncontrolled production of reactive oxygen species and lipid peroxidation. These features are also common to ferroptosis, an iron-mediated type of cell death triggered by accumulation of lipoperoxides with distinct morphological and molecular characteristics with respect to other known cell deaths. Scope of review: Even though ferroptosis has been associated with various neurodegenerative diseases including FRDA, the mechanisms leading to disease onset/progression have not been demonstrated yet. We describe the molecular alterations occurring in FRDA that overlap with those characterizing ferroptosis. Major conclusions: The study of ferroptotic pathways is necessary for the understanding of FRDA pathogenesis, and anti-ferroptotic drugs could be envisaged as therapeutic strategies to cure FRDA.

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Somatic instability of the expanded GAA repeats in Friedreich’s ataxia

PLoS ONE ◽

10.1371/journal.pone.0189990 ◽

2017 ◽

Vol 12 (12) ◽

pp. e0189990 ◽

Cited By ~ 22

Author(s):

Ashlee Long ◽

Jill S. Napierala ◽

Urszula Polak ◽

Lauren Hauser ◽

Arnulf H. Koeppen ◽

...

Keyword(s):

Friedreich’S Ataxia ◽

Friedreich's Ataxia ◽

Somatic Instability ◽

Gaa Repeats

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Interruptions of the FXN GAA Repeat Tract Delay the Age at Onset of Friedreich’s Ataxia in a Location Dependent Manner

International Journal of Molecular Sciences ◽

10.3390/ijms22147507 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7507

Author(s):

Suran Nethisinghe ◽

Maheswaran Kesavan ◽

Heather Ging ◽

Robyn Labrum ◽

James M. Polke ◽

...

Keyword(s):

Trinucleotide Repeat ◽

Age At Onset ◽

Disease Onset ◽

Friedreich’S Ataxia ◽

Friedreich's Ataxia ◽

Dependent Manner ◽

Repeat Tract ◽

Delay Disease Onset ◽

Intron 1 ◽

Exponential Decay Model

Friedreich’s ataxia (FRDA) is a comparatively rare autosomal recessive neurological disorder primarily caused by the homozygous expansion of a GAA trinucleotide repeat in intron 1 of the FXN gene. The repeat expansion causes gene silencing that results in deficiency of the frataxin protein leading to mitochondrial dysfunction, oxidative stress and cell death. The GAA repeat tract in some cases may be impure with sequence variations called interruptions. It has previously been observed that large interruptions of the GAA repeat tract, determined by abnormal MboII digestion, are very rare. Here we have used triplet repeat primed PCR (TP PCR) assays to identify small interruptions at the 5′ and 3′ ends of the GAA repeat tract through alterations in the electropherogram trace signal. We found that contrary to large interruptions, small interruptions are more common, with 3′ interruptions being most frequent. Based on detection of interruptions by TP PCR assay, the patient cohort (n = 101) was stratified into four groups: 5′ interruption, 3′ interruption, both 5′ and 3′ interruptions or lacking interruption. Those patients with 3′ interruptions were associated with shorter GAA1 repeat tracts and later ages at disease onset. The age at disease onset was modelled by a group-specific exponential decay model. Based on this modelling, a 3′ interruption is predicted to delay disease onset by approximately 9 years relative to those lacking 5′ and 3′ interruptions. This highlights the key role of interruptions at the 3′ end of the GAA repeat tract in modulating the disease phenotype and its impact on prognosis for the patient.

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