Genome compartmentalization is linked to the replication program and mutational outcomes in Trypanosoma cruzi

DNA replication dynamics are important contributors to genome variability across eukaryotes, including protozoan systems. Trypanosoma cruzi is a unique model, as its genome contains Core and Disruptive compartments with distinct gene content and evolutionary behavior. To understand how replication shapes genome variation, we applied new analytical frameworks to our D-NAscent dataset to map replication events genome-wide at base-pair resolution, using replication fork directionality and density. Low-activity replication sites, such as low-efficiency origins and sporadic termination events, occur more frequently in the conserved Core compartment, and are associated with SNPs rather than loss of heterozygosity (LOH). In contrast, highly active sites, such as efficient origins and recurrent termination regions, are found in rapidly evolving compartments, where they accumulate more SNPs and are associated with higher LOH. Notably, low-efficiency origins generate more SNPs in regions prone to head-on transcription conflicts, whereas efficient origins tend to accumulate SNPs mainly in co-directional contexts. Recurrent termination sites are enriched downstream of origins, regardless of transcriptional orientation. Collectively, these findings indicate that replication activity levels predict mutational outcomes and unveil compartment-specific replication programs that shape genome stability and diversification in T. cruzi .