Takacs, Z.; Mylarshchikov, D.; Kolesnikova, S.; Langer, C. C. H.; Goloborodko, A.; Gerlich, D. W.

During each cell cycle, cells must not only duplicate DNA sequence but also preserve the three-dimensional genome architecture required for gene regulation and DNA repair. After replication, this requires coordination between cohesin-mediated loop extrusion and sister-chromatid cohesion for homology-directed DNA repair. Because extrusion promotes sister resolution whereas cohesion can impede extrusion, how both activities coexist on replicated chromosomes is unclear. Here we show that cohesin function is partitioned within the nested TAD hierarchy: cohesive cohesin accumulates at high-level boundaries to form cohesion zones, while loop-extruding cohesin occupies boundaries across all hierarchical levels. Our data support a model in which this segregation emerges from dynamic interplay between cohesin pools and semi-permeable CTCF barriers, which act in cis and in trans to constrain sister-sister misalignment. Thus, a CTCF-defined boundary framework enables sister tethering while preserving dynamic loop folding, allowing replicated genomes to simultaneously support gene regulation and faithful DNA repair.