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.

• Keywords
• Nup88, Nup98 condensates, Sec13, double-strand break repair, droplets, heterochromatin repair, homologous recombination, nuclear dynamics, nucleoplasmic nucleoporins, phase separation,
• MeSH
• Chromosomal Proteins, Non-Histone metabolism   genetics   MeSH
• Drosophila melanogaster * genetics   metabolism   MeSH
• DNA Breaks, Double-Stranded MeSH
• Heterochromatin * genetics   metabolism   MeSH
• Chromobox Protein Homolog 5 MeSH
• Nuclear Pore Complex Proteins * metabolism   genetics   MeSH
• Cell Cycle Proteins metabolism   genetics   MeSH
• Drosophila Proteins * metabolism   genetics   MeSH
• Recombinational DNA Repair * MeSH
• Rad51 Recombinase * metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromosomal Proteins, Non-Histone MeSH
• Heterochromatin * MeSH
• Chromobox Protein Homolog 5 MeSH
• Nuclear Pore Complex Proteins * MeSH
• nuclear pore complex protein 98 MeSH Browser
• Cell Cycle Proteins MeSH
• Drosophila Proteins * MeSH
• Rad51 Recombinase * MeSH

The intrinsically disordered carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNAPII) consists of multiple tandem repeats of the consensus heptapeptide Y1-S2-P3-T4-S5-P6-S7. The CTD promotes liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the role of the conserved heptad residues in LLPS is hampered by the lack of direct biochemical characterization of the CTD. Here, we generated a systematic array of CTD variants to unravel the sequence-encoded molecular grammar underlying the LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of tyrosine and cis-trans isomerization of prolines govern CTD phase-separation. The cis conformation of prolines and β-turns in the SPXX motif contribute to a more compact CTD ensemble, enhancing interactions among CTD residues. We further demonstrate that prolines and tyrosine in the CTD consensus sequence are required for phosphorylation by Cyclin-dependent kinase 7 (CDK7). Under phase-separation conditions, CDK7 associates with the surface of the CTD droplets, drastically accelerating phosphorylation and promoting the release of hyperphosphorylated CTD from the droplets. Our results highlight the importance of conformationally restricted local structures within spacer regions, separating uniformly spaced tyrosine stickers of the CTD heptads, which are required for CTD phase-separation.

• MeSH
• Cyclin-Dependent Kinases * metabolism   chemistry   MeSH
• Phosphorylation MeSH
• Cyclin-Dependent Kinase-Activating Kinase * MeSH
• Humans MeSH
• Proline metabolism   chemistry   MeSH
• Protein Domains MeSH
• RNA Polymerase II * metabolism   chemistry   MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation * MeSH
• Tyrosine metabolism   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDK7 protein, human MeSH Browser
• Cyclin-Dependent Kinases * MeSH
• Cyclin-Dependent Kinase-Activating Kinase * MeSH
• Proline MeSH
• RNA Polymerase II * MeSH
• Tyrosine MeSH