Reconstitution of replication stalling by triplex forming repeats
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Embargo End Date
2026-10-28
ICR Authors
Authors
Sabin, F
Document Type
Thesis or Dissertation
Date
2026-04-28
Date Accepted
Abstract
Repetitive DNA sequences capable of forming secondary structures, including triplexes, G-quadruplexes, i-motifs, hairpins and cruciforms, serve functional roles in processes such as gene regulation and chromatin organisation. However, it has become clear that secondary DNA structures can also be potent sources of genome instability and are strongly implicated in numerous human diseases. Expansion of an intronic triplex-forming (GAA/TTC)n repeat causes Friedreich's ataxia, the most common inherited ataxia. Recently, triplex-forming repeats have been implicated in cancer, where they cause significant chromosomal fragility and repeat instability. Importantly, triplex-forming repeats stall DNA replication in vivo which results in chromosomal fragility. Furthermore, many replication factors are crucial for maintaining repeat stability. Despite the well-established association of replication with chromosomal fragility and repeat instability, a detailed mechanistic understanding of this relationship is still missing.In this work, I reconstitute DNA replication stalling by triplex-forming repeats using the in vitro budding yeast replication system. I show that expanded (GAA/TTC)n repeats stall DNA replication forks in either repeat orientation relative to the origin. In addition, I uncover a novel lagging strand-specific defect that occurs only when (TTC)n or (T)n serves as the lagging strand template. I show that the lagging strand defect at (TTC)n repeats is likely caused by the generation of long Okazaki fragment flaps that cannot be processed by Fen1 but instead require Dna2 for their processing. I also explore the contribution of triplex formation at these repeats by using 7-deaza-purine dNTP analogues and by making small mutations within (GAA/TTC)n repeats that disrupt triplex-forming potential. Finally, I systematically test the ability of a broad range of helicases implicated in the unwinding of triplexes to rescue DNA replication stalling. Altogether, this work provides mechanistic insight into the replication dynamics of triplex-forming repeats.
Citation
2026
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Genome Replication