Friedreich's ataxia–associated GAA repeats induce replication-fork reversal and unusual molecular junctions

Abstract

Expansion of GAA/TTC repeats is the causative event in Friedreich's ataxia. GAA repeats have been shown to hinder replication in model systems, but the mechanisms of replication interference and expansion in human cells remained elusive. To study in vivo replication structures at GAA repeats, we designed a new plasmid-based system that permits the analysis of human replication intermediates by two-dimensional gel electrophoresis and EM. We found that replication forks transiently pause and reverse at long GAA/TTC tracts in both orientations. Furthermore, we identified replication-associated intramolecular junctions, located between GAA/TTC repeats and other homopurine-homopyrimidine tracts, that were associated with breakage of the plasmid fork not traversing the repeats. Finally, we detected postreplicative, sister-chromatid hemicatenanes on control plasmids, which were replaced by persistent homology-driven junctions at GAA/TTC repeats. These data prove that GAA/TTC tracts interfere with replication in humans and implicate postreplicative mechanisms in trinucleotide repeat expansion.

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

“…7). This is in agreement with the recent finding that repetitive DNA sequences with a propensity to form non-B DNA structures induce reversal of traversing replication forks at remarkably high frequencies (29). Considering the high number of endogenous DNA lesions (30) and the abundance of non-B-forming structures in the human genome (31), it is conceivable that fine-tuning of PAR synthesis and degradation at a number of chromosomal locations plays a pivotal role in assisting complete and faithful replication of the human genome.…”

“…Considering the high number of endogenous DNA lesions (30) and the abundance of non-B-forming structures in the human genome (31), it is conceivable that fine-tuning of PAR synthesis and degradation at a number of chromosomal locations plays a pivotal role in assisting complete and faithful replication of the human genome. These observations may contribute to explain the mitotic defects previously associated with the essential role of PARG in development, as deletion of all isoforms of PARG leads to embryonic lethality in mice (29).…”

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability

“…We propose that some stalled forks, especially for longer CAG-130 repeats, are not able to proceed; in this case, the replisome may be dissociated or modified to create a "collapsed" fork with or without a DNA break. Fork reversal may occur, as has been proposed (Mirkin 2006) and shown for CAG and GAA triplet repeats (Fouche et al 2006;Kerrest et al 2009;Follonier et al 2013). Note that a lagging strand hairpin is shown initiating the fork reversal, but it could also occur by a leading strand hairpin; in this case, replication through some of the repeats would be needed for the repeat sequence to be part of the regressed arm of the fork.…”

Replication fork instability and the consequences of fork collisions from rereplication

“…Fork reversal was long thought to be the result of failed checkpoint response to fork stalling, but a recent study in human cell culture demonstrated that fork reversal is a common response to various replication perturbations when the checkpoint is intact (Zellweger et al 2015). Fork reversal also is observed at trinucleotide repeats (Follonier et al 2013), suggesting that chicken foot structures can form during unperturbed replication at hard to replicate sequences. Formation of reversed forks is dependent on PARP-1 regulation of the RECQ1 helicase as well as Rad51 (Zellweger et al 2015).…”

“…Replication forks frequently stall at these AT repeats and lead to DNA breaks in the absence of replication stress, and stalling is enhanced in the presence of aphidicolin (Ozeri-Galai et al 2012). Secondary structures formed by trinucleotide repeats cause fork pausing and reversal and frequently correspond to break formation (Follonier et al 2013;Liu et al 2013;Gerhardt et al 2014). One extensively studied example is G quadruplexes (G4s), highly stable secondary structures that form at stretches of G-rich DNA.…”