Crosstalk at crossroads: Maintaining fork integrity and genome stability under replication stress

DNA replication is essential for genome stability, yet replication forks frequently encounter obstacles that induce replication stress. If not properly stabilized or restarted, stalled forks can lead to genome instability, a hallmark of cancer. This thesis investigates the molecular mechanisms that protect replication forks under stress and their relevance to tumorigenesis and therapy response. Using a proteomics-based approach, we identify RIF1 as a key replication fork protection factor. Independently of homologous recombination, RIF1 safeguards stalled forks by restraining DNA2 nuclease activity through its interaction with Protein Phosphatase 1. Loss of RIF1 results in delayed fork restart, under-replicated DNA, and genome instability, revealing a novel fork protection pathway. This thesis further highlights the functional separation between replication fork protection and DNA double-strand break repair, particularly in the context of BRCA proteins. Modelling a patient-derived BRCA1 variant that disrupts BRCA1–PALB2 interaction demonstrates that fork protection can be preserved despite homologous recombination defi ciency, with important implications for chemotherapeutic response. Finally, this thesis identifi es ELOF1 as a critical factor in resolving transcription–replication confl icts by promoting transcription-coupled nucleotide excision repair and enabling replication fork progression following UV damage. Loss of ELOF1 leads to replication stress and genome instability. Together, these fi ndings advance our understanding of replication fork protection mechanisms and their roles in maintaining genome stability, tumorigenesis, and therapeutic response.