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 -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.