Transcription directs the migration of crossover-designated Holliday junctions from Spo11 hotspots toward cohesin-associated chromosome axis sites at convergent transcription regions, where crossover resolution preferentially occurs, thereby coordinating the molecular events of meiotic recombination with chromosome architecture to ensure accurate chromosome segregation.

During meiosis, genetic diversity arises from the resolution of branched DNA intermediates called Holliday junctions to create crossovers—sites of reciprocal exchange between parental chromosomes. Holliday junctions arise during the repair of Spo11-induced DNA breaks, yet the principles linking break formation, repair, and chromosome architecture remain unclear. Top3, a type IA topoisomerase, acts on Holliday junctions, but its spatiotemporal dynamics are unknown. Here, we map Top3 catalytic activity throughout meiotic prophase with strand specificity and nucleotide resolution. We identify a DNA sequence motif associated with catalysis and a pattern of activity around Spo11 hotspots that requires ongoing repair and Top3’s helicase partner, Sgs1. Strikingly, Top3 activity shifts over time, influenced by transcription, cohesin, and the crossover factors Msh5 and Mer3, redistributing toward sites of convergent transcription—known locations of meiotic cohesin association. Upon prophase exit, as crossovers are resolved, Top3 activity subsides. Remarkably, maps of genome-wide recombination reveal that crossover resolution preferentially occurs at these same regions of convergent transcription. Collectively, we propose that Top3 coordinates the transcription-coupled movement of Holliday junctions from Spo11 hotspots towards cohesin-associated axis sites, whereupon crossover resolution occurs to ensure accurate meiotic chromosome segregation.