Mechanisms of fork stalling and reversal at pathogenic CANVAS (AAGGG)n repeats
Abstract
Expansions of the pathogenic (AAGGG)n repeat within the RFC1 gene cause the hereditary disease CANVAS. We have shown that this repeat impedes DNA replication both in vitro and in vivo. Here, we investigated the mechanisms of replication fork stalling and reversal at (AAGGG)n repeats within an SV40-based episome in human cells. A comprehensive 2-dimensional electrophoretic analysis of replication intermediates revealed a strict orientation dependence of the replication block, evident only when the homopurine sequence was in the lagging-strand template. Fork accumulation was further increased in the presence of the G4-DNA stabilizer pyridostatin, implicating G4-DNA in the process. This stalling was followed by an accumulation of reversed forks and further analysis revealed roles for SMARCAL1 and RAD51 in this process. Biochemical studies showed that SMARCAL1, with assistance from RPA, efficiently remodels a fork carrying the G4-forming AAGGG repeat in the ssDNA gap, but not a substrate carrying the repeat in triplex conformation. We conclude that stalling results from G4-DNA formation by the repeat, which then triggers SMARCAL1-mediated fork reversal at the repeat. Given the mirror symmetry of the sequence, the nascent strands at the reversed forks likely adopt a triplex conformation, leading to a thorough accumulation of branched structures.
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BibTeX
@misc{mandel2026mechanisms,
title = {{Mechanisms of fork stalling and reversal at pathogenic CANVAS (AAGGG)n repeats}},
author = {Nicholas H. Mandel and Sofia Movafaghi and Rishika Vaid and Olga Usalka and Simone Ummarino and Joe M. Polyak and Masayoshi Honda and Negin Mozafari and Rula Zain and Maria Spies and Sergei M. Mirkin},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.27.762263},
url = {https://www.biorxiv.org/content/10.64898/2026.10.27.762263v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Mechanisms of fork stalling and reversal at pathogenic CANVAS (AAGGG)n repeats
AU - Nicholas H. Mandel
AU - Sofia Movafaghi
AU - Rishika Vaid
AU - Olga Usalka
AU - Simone Ummarino
AU - Joe M. Polyak
AU - Masayoshi Honda
AU - Negin Mozafari
AU - Rula Zain
AU - Maria Spies
AU - Sergei M. Mirkin
PY - 2026
DA - 2026/10/27
PB - bioRxiv
DO - 10.64898/2026.10.27.762263
UR - https://www.biorxiv.org/content/10.64898/2026.10.27.762263v1
AB - Expansions of the pathogenic (AAGGG)n repeat within the RFC1 gene cause the hereditary disease CANVAS. We have shown that this repeat impedes DNA replication both in vitro and in vivo. Here, we investigated the mechanisms of replication fork stalling and reversal at (AAGGG)n repeats within an SV40-based episome in human cells. A comprehensive 2-dimensional electrophoretic analysis of replication intermediates revealed a strict orientation dependence of the replication block, evident only when the homopurine sequence was in the lagging-strand template. Fork accumulation was further increased in the presence of the G4-DNA stabilizer pyridostatin, implicating G4-DNA in the process. This stalling was followed by an accumulation of reversed forks and further analysis revealed roles for SMARCAL1 and RAD51 in this process. Biochemical studies showed that SMARCAL1, with assistance from RPA, efficiently remodels a fork carrying the G4-forming AAGGG repeat in the ssDNA gap, but not a substrate carrying the repeat in triplex conformation. We conclude that stalling results from G4-DNA formation by the repeat, which then triggers SMARCAL1-mediated fork reversal at the repeat. Given the mirror symmetry of the sequence, the nascent strands at the reversed forks likely adopt a triplex conformation, leading to a thorough accumulation of branched structures.
SN - 2692-8205
ER -