The co-synthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences are still little studied. Here, we showed that the number of constitutive origins mapped in the Trypanosoma brucei genome is less than the minimum required to complete replication within S-phase duration. By the development of a mechanistic model of DNA replication considering replication-transcription conflicts and using immunofluorescence assays and DNA combing approaches, we demonstrated that the activation of non-constitutive (backup) origins are indispensable for replication to be completed within S-phase period. Together, our findings suggest that transcription activity during S phase generates R-loops, which contributes to the emergence of DNA lesions, leading to the firing of backup origins that help maintain robustness in S-phase duration. The usage of this increased pool of origins, contributing to the maintenance of DNA replication, seems to be of paramount importance for the survival of this parasite that affects million people around the world.

The cosynthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences have not yet been investigated. Here, using equations and computational analysis we demonstrated that the number of constitutive origins mapped in the Trypanosoma brucei genome is close to the minimum required to complete replication within S phase duration. However, taking into account the location of these origins in the genome, the replication in due time becomes virtually impossible, making it necessary to activate non-constitutive origins. Moreover, computational and biological assays pointed to transcription being responsible for activating non-constitutive origins. Together, our results suggest that transcription action through conflicts with replication contributes to the firing of non-constitutive origins, maintaining the robustness of S phase duration. The usage of this entire pool of origins seems to be of paramount importance for the survival of this parasite that infects million people around the world since it contributes to the maintenance of the replication of its DNA.