Cohesin–axis interaction via a conserved Red1 motif promotes domain-specific DSB formation and Mek1 activation

Faithful chromosome segregation during meiosis I requires tight control of interhomolog recombination. In budding yeast, the meiotic chromosome axis, built on Rec8-containing cohesin together with Red1 and Hop1, acts as a central platform regulating meiotic recombination from programmed DNA double-strand break (DSB) formation to checkpoint signaling and chromosome segregation, yet how cohesin recruits axis proteins remains unclear. Here, we identified a conserved cohesin-interacting motif (CIM) in Red1 that directly binds Rec8. AlphaFold3 modeling predicted that Red1-CIM forms a short α-helix that docks into a conserved hydrophobic pocket within the Rec8 C-terminal winged-helix domain, which we confirmed biochemically. Disruption of the Red1-CIM preferentially impaired Red1 recruitment to Rec8-dependent chromosomal regions, while relative enrichment in Rec8-independent domains was preserved, leading to reduced DSB formation in Rec8-dependent domains. The Red1-CIM mutation also reduced crossover formation, increased chromosome missegregation, and reduced spore viability. Notably, this spore lethality exceeded that predicted by the reduction in DSB formation. Consistently, red1-CIM mutants failed to activate the meiotic checkpoint kinase Mek1. Finally, we provide evolutionary, structural, and biochemical evidence that this Red1–Rec8 interaction is conserved across fungi and plants. Together, these findings define a direct molecular bridge linking cohesin to chromosome-axis organization, spatial DSB regulation, and checkpoint signaling during meiosis.