Fig. 7: TRF1’s capacity to reduce telomere fragility depends on fork reversal, PrimPol activity, and RNA-DNA hybrids.

A Lagging (green) and leading (black) telomere fragility assessed by CO-FISH in PCS^LT-EV, PCS^LT-TRF1 or PCS^LT-TRF1-RH1 cells under the specified conditions. siSM1: siSMARCAL1. Cells were treated with 60 µM spironolactone (SP), or DMSO as control, during 25 h before BrdU/BrdC incorporation. A minimum of 5900 chromosome ends were analyzed, based on n metaphase spreads collected from three independent biological replicates. Boxplot: Min, 1st quartile, Median, 3rd quartile, and Max. Kruskal–Wallis tests. Source data are provided as a Source Data file. B Model. Stalled replication forks at PCS^LT telomeres undergo SMARCAL1-dependent reversal, generating new 3′ telomeric ends. The reversed forks are bound by telomerase. Fork replication can resume through a homologous recombination-dependent restart process that requires RAD51 and might be facilitated by long 3′ overhangs generated by the telomerase and the formation of RNA:DNA hybrid formation, possibly involving TFIIH. If reversal is suppressed, PrimPol-dependent repriming can promote fork restart. This process may also be dependent on RNA:DNA hybrids. Optimal levels of TRF1 are required to suppress telomere fragility, by facilitating the reversal/restart processing of stalled forks. Protein illustrations were generated using BioRender (Vaurs, M. (2025) https://BioRender.com/ke04c93).