Drosophila Nse5/6: a missing link for heterochromatin repair

SUMMARY DSBs occur in every cell as a result of cell exposure to environmental mutagens, such as ionizing radiation, or during normal cell metabolism, such as DNA replication. Repairing DSBs is particularly challenging in pericentromeric heterochromatin, a poorly characterized domain in which the abundance of repeated sequences can trigger widespread aberrant recombination and genome instability. We have previously identified a specialized and conserved pathway that promotes faithful homologous recombination (HR) repair of heterochromatin, while preventing aberrant recombination. HR starts inside the heterochromatin domain with resection, but it continues only after a striking relocalization of repair sites to the nuclear periphery. Relocalization likely prevents aberrant recombination by isolating DSBs and their repair templates (on the sister chromatid or homologous chromosome) away from similar sequences on other chromosomes, before strand invasion. A key regulator of this pathway is the Smc5/6 complex that halts HR repair inside the heterochromatin domain through SUMOylation, and promotes DSB mobilization in mysterious ways. Nse5/6 is a linchpin regulator of this complex that mediates Smc5/6 targeting to DSBs while inhibiting its ATP-ase and loop extrusion functions. Despite this critical role, Nse5/6 is an elusive component of the Smc5/6 complex. This rapidly evolving heterodimer is poorly conserved at the sequence level, and it has only been identified in yeasts, mammalian cells, and Arabidopsis. Drosophila cells are currently the best model system to study heterochromatin repair dynamics, but the lack of known Nse5/6 subunits limits our understanding of Smc5/6 regulation in this context. With the overarching goal of illuminating the mechanisms of heterochromatin repair, the objective of this proposal is the identification and characterization of Drosophila Nse5/6, and its functions in heterochromatin repair. We identified strong candidates to perform Nse5/6 functions in Drosophila through a mass spectrometry screening for Smc5/6 interactions and molecular modeling. We will combine structural determination at the atomic level with targeted mutagenesis and assays in vitro and in vivo to identify this missing link in genome stability. Expected positive outcomes of this research include the first identification and characterization of Drosophila Nse5/6 and its functional domains. In addition, we will define new mechanisms specifically targeting Nse5/6 to heterochromatin and repair sites, a novel regulatory function relying on its phosphorylation, and the establishment of its role(s) in heterochromatin repair. These results will have an important positive impact by identifying crucial safeguard mechanisms used by normal cells to protect the genome from environmental threats. These studies are also expected to produce new Drosophila disease models, and to significantly advance our understanding of human diseases resulting from Nse5/6 dysregulation, including cancer, neurodevelopmental disorders, and viral infection.