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SUMMARY Phase separation forms membraneless compartments in the nuclei, including by establishing heterochromatin “domains” and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination, and “safe” homologous recombination (HR) repair of these sequences requires the movement of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation to these dynamics is unclear. Here, we show that Nup98 nucleoporin is recruited to heterochromatic repair sites before relocalization through Sec13 or Nup88 nucleoporins, and downstream from the Smc5/6 complex and SUMOylation. Remarkably, the phase separation properties of Nup98 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 a novel “off-pore” role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote. HighlightsNup88 and Sec13 recruit Nup98 to heterochromatic DSBs downstream from Smc5/6Nup88, Sec13 and Nup98 promote repair focus mobilization in heterochromatin ‘off-pore’Nup98 excludes Rad51 from repair sites inside the heterochromatin domainPhase separation by Nup98 is required and sufficient for relocalization and Rad51 exclusion
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Alternative end-joining results in smaller deletions in heterochromatin relative to euchromatin
Pericentromeric heterochromatin is highly enriched for repetitive sequences prone to aberrant recombination. Previous studies showed that homologous recombination (HR) repair is uniquely regulated in this domain to enable ‘safe’ repair while preventing aberrant recombination. In Drosophila cells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR repair. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how alternative EJ (alt-EJ) pathways operate in heterochromatin remains largely uncharacterized. Here, we induce DSBs in single euchromatic and heterochromatic sites using a new system that combines the DR-white reporter and I-SceI expression in spermatogonia of flies. Using this approach, we detect higher frequency of HR repair in heterochromatin, relative to euchromatin. Further, sequencing of mutagenic repair junctions reveals the preferential use of different EJ pathways across distinct euchromatic and heterochromatic sites. Interestingly, synthesis-dependent microhomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin.
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- Award ID(s):
- 1751197
- PAR ID:
- 10556311
- Publisher / Repository:
- eLife
- Date Published:
- Journal Name:
- eLife
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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