Phase separation forms membraneless compartments, including heterochromatin "domains" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination. In Drosophila cells, "safe" homologous recombination (HR) repair of these sequences requires their relocalization to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation is unclear. Here, we show that Nup98 nucleoporin is recruited to repair sites before relocalization by Sec13 or Nup88, and downstream of the Smc5/6 complex and heterochromatin protein 1 (HP1). Remarkably, Nup98 condensates are immiscible with HP1 condensates, and they are required and sufficient to mobilize repair sites and exclude Rad51, thus preventing aberrant recombination while promoting HR repair. Disrupting this pathway results in heterochromatin repair defects and widespread chromosome rearrangements, revealing an "off-pore" role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote.

Department of Biochemistry and Structural Biology and Greehey Children's Cancer Research Institute University of Texas Health Science Center at San Antonio San Antonio TX 78229 USA

Department of Biological Sciences Molecular and Computational Biology Section University of Southern California Los Angeles CA 90089 USA

Department of Biological Sciences Molecular and Computational Biology Section University of Southern California Los Angeles CA 90089 USA; Department of Biochemistry and Molecular Biology Cumming School of Medicine University of Calgary Calgary AB T2N 4N1 Canada

Department of Biological Sciences Molecular and Computational Biology Section University of Southern California Los Angeles CA 90089 USA; Department of Genetics Harvard Medical School Boston MA 02115 USA

Department of Biology Faculty of Medicine Masaryk University Kamenice 5 A7 Brno 62500 Czech Republic

Department of Biology Faculty of Medicine Masaryk University Kamenice 5 A7 Brno 62500 Czech Republic; National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 A4 62500 Brno Czech Republic

Department of Biology San Diego State University San Diego CA 92182 USA

Department of Chemistry University of Pennsylvania Philadelphia PA 19104 USA

Erratum In

Mol Cell. 2025 Aug 14:S1097-2765(25)00662-8. doi: 10.1016/j.molcel.2025.08.012. PubMed

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