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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This content will become publicly available on December 8, 2024
‘Off-pore’ nucleoporins relocalize heterochromatic breaks through phase separation
Heterochromatin mostly comprises repeated DNA sequences prone to ectopic recombination. In Drosophila cells, ‘safe’ homologous recombination (HR) repair of heterochromatic double-strand breaks (DSBs) requires relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. Relocalization is driven by nuclear actin filaments and myosins, while anchoring is mediated by the Nup107 complex at nuclear pores. Here, we show an additional ‘off pore’ role of nucleoporins in heterochromatin repair. Sec13 and Nup88 independently recruit Nup98 to DSBs before relocalization and downstream from the Smc5/6 complex and SUMOylation. Remarkably, the phase separation properties of Nup98 are required and sufficient to induce the mobilization of repair sites and to exclude Rad51, thus preventing aberrant recombination while facilitating HR repair. Disrupting this pathway results in heterochromatin repair defects, revealing a novel role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote.
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- Award ID(s):
- 1751197
- NSF-PAR ID:
- 10484129
- Publisher / Repository:
- Biorixv
- Date Published:
- Journal Name:
- Biorxiv
- ISSN:
- 2692-8205
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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