Abstract
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.
Highlights
DNA replication is an essential tightly regulated process, basis of genetic inheritance, which follows a specific temporal program
After applying this formula to each chromosome, we compared the minimum number of origins obtained with the number of constitutive origins mapped by the marker frequency analysis coupled with deep sequencing (MFA-seq) technique, and we found that these values were very similar
Interesting, in the same study in which v = 1.84 kb.min−1 was estimated, the authors suggested an average inter-origin distance (IOD) of 148.8 kb, i.e., one origin per ~148.8 kb. If we extrapolate this to all eleven chromosomes, we find an average number of origins per chromosome that is 3.1 times higher than the mo estimated with v = 1.84 kb.min−1, 4.5 times higher than the constitutive origins estimated by MFA-seq, and 5.9 times higher than the mo estimated with v = 3.7 kb.min−1 (Fig. 2B)
Summary
DNA replication is an essential tightly regulated process, basis of genetic inheritance, which follows a specific temporal program. We designed and simulated a mechanistic model of DNA replication considering origin firing in the presence or absence of polycistronic transcription during S phase These simulations suggested robustness in S-phase duration relative to increasing levels of transcriptional activity, which were compensated by increased origin firing. Based on these results, we hypothesize that if there is transcription during S phase, the co-synthesis of DNA and RNA can generate collisions between the two associated types of machinery, activating non-constitutive (backup) origins and increasing the pool of origins used to complete S phase. Our findings suggest that the action of the transcription machinery (probably through conflicts with replication) contributes to the activation of backup origins helping to maintain robustness in S-phase duration in T. brucei, a human pathogen of great medical importance
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