By revealing that transcriptional memory directs meiotic breaks in essential genes toward rapid non-crossover repair, this work overturns the assumption that crossover placement is break-driven and establishes a conserved principle protecting gene integrity across mammals.
Meiotic DNA double-strand breaks segregate into two temporally distinct repair modes—fast-resolving breaks that are overwhelmingly repaired as non-crossovers and slow-resolving breaks that generate nearly all crossovers—with repair fate determined by transcriptional marking of genes in early meiosis (zygotene) through H3K36me3 chromatin modifications and chromosome axis architecture, thereby directing crossovers away from a conserved set of ~4,500 transcriptionally critical genes across mammalian species and safeguarding their function while shaping genome evolution.
Crossovers are essential for accurate chromosome segregation in meiosis. Yet the programmed DNA double-strand breaks that initiate them frequently occur in genes and pose a risk to transcription required for gametogenesis. How meiotic cells reconcile these competing demands has remained unclear. Here, we generate a genome-wide in vivo atlas of meiotic recombination intermediates across ∼42,000 hotspots by mapping repair proteins BLM, HFM1, and RPA in wild-type and genome-engineered mutant mouse testes. These maps reveal two distinct modes of break repair: a fast-resolving class with short-lived intermediates that are repaired predominantly as non-crossovers, and a slower class with persistent intermediates that give rise to nearly all crossovers. Fast-resolving hotspots occur almost exclusively within a deeply conserved set of ∼4,500 genes marked by structural and chromatin features established during an early stage of meiotic transcription. This transcriptional memory predicts repair fate with high accuracy across mouse subspecies and sexes. Across widely diverged mammals, including humans and cattle, orthologous genes show similar crossover suppression. Our findings reveal an early bifurcation between crossover and non-crossover repair that is governed by the transcriptional context of meiotic breaks. Together, they establish an evolutionarily conserved principle in which crossovers are directed away from transcriptionally important genes, thereby safeguarding gene function and shaping their evolution.