Jessica Spindler, Francesca Pandolfo, Anna Eva Koch, Priscilla Piccirillo, Drew Jordahl, Nikhil Venkatesh, Dhruthi Suresh, K R Ylvisaker, Anita Jopkiewicz, Johanna Bihler, Sandra Buschbaum, Marcel Morgenstern, Katherine A. Overmyer, Estelle Vincendeau, Joshua J. Coon, Pei‐Chi Wei, Robert Hänsel‐Hertsch, Kavi P M Mehta, Stephanie Panier
Faithful DNA replication is essential for genome stability, yet replication forks face constant stress. The Bloom syndrome helicase (BLM) safeguards fork integrity, but excessive BLM activity can itself induce replication stress. We identify SLX4IP as a genome-wide regulator that restrains BLM to maintain replication fork stability. SLX4IP localizes broadly across chromatin with recruitment enhanced under replication stress. Loss of SLX4IP slows replication forks, remodels the replisome, and generates post-replicative single-stranded DNA gaps that are accompanied by elevated nuclear ADP ribose, reflecting compromised replication integrity. These defects are driven by dysregulated BLM activity, establishing SLX4IP as a negative regulator of BLM-dependent replication stress. At ALT telomeres, SLX4IP deficiency triggers ATR signaling, telomere fragility, and accumulation of ALT-associated PML bodies. Here, SLX4IP functions in parallel with FANCM to restrain BLM at ALT telomeres, with co-depletion of SLX4IP and FANCM causing synthetic lethality in ALT-positive cells, a phenotype fully rescued by BLM loss. Together, our results define SLX4IP as a critical genome-wide regulator of replication fork integrity and reveal SLX4IP as a potential vulnerability in ALT-positive cancers.