Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration

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.

Widespread roles ofTrypanosoma bruceiATR in nuclear genome function and transmission are linked to R-loops

“…The organisation of the T. brucei genome into ∼200 multigene transcription units 86 , some containing hundreds of co-transcribed genes, might be interpreted as the parasite only encoding large genes, with the prediction of transcription-replication conflicts being prevalent and localised. Indeed, transcription is observed throughout the T. brucei cell cycle 87 , and mapping suggests DNA replication forks meeting multigene transcription head-on are slowed relative to those that are co-directional with transcription 88 . Despite these observations, previous mapping, with and without loss of RNase H1 or RNase H2, did not reveal accumulation of R-loops at predictable locations relative to origins 52 , and no such pattern was detected here after TbATR RNAi.…”

Comparative Analysis of the Minimum Number of Replication Origins in Trypanosomatids and Yeasts

“…The replication time required for all chromosomes determines the S-phase duration. Although the S-phase length is referred to as a way of regulating the cell cycle progression [4,5], recent studies have suggested that it is extremely robust [6][7][8].…”

“…The exact number of origins per chromosome can vary according to cell type and the cellular environment [14]. However, the minimum number of origins (MO) required to duplicate an entire chromosome within a specific S-phase duration must show minimal variation because it depends on two very constant factors: average replication rate and chromosome size [8].…”

“…In trypanosomatids, single-celled eukaryotes that encompass human pathogens are of great medical importance, and the question about how many origins are needed to replicate an entire chromosome during the S-phase is totally open [8,10,15,16]. Even for the widely studied domain Bacteria and the model eukaryote S. cerevisiae, this discussion is not yet a closed subject [1,[17][18][19].…”

A DOT1B/Ribonuclease H2 protein complex is involved in R-loop processing, genomic integrity and antigenic variation in Trypanosoma brucei

“…Different possibilities for a source of recombination-initiating DNA breaks have been proposed, and a recently published model has suggested the participation of RNA-DNA hybrids (so-called R-loops) (10). R-loops in T. brucei are generated by transcription (11), and usually removed by the two types of Ribonuclease H (RH1 and RH2) (12)(13)(14). DNA lesions and altered VSG expression were observed if R-loops were not processed properly (12,13), or if their formation at the ES was not accurately coordinated by the telomere-associated protein RAP1 (15).…”