Recruitment kinetics of the homologous recombination pathway in procyclic forms of Trypanosoma brucei after ionizing radiation treatment

Abstract

One of the most important mechanisms for repairing double-strand breaks (DSBs) in model eukaryotes is homologous recombination (HR). Although the genes involved in HR have been found in Trypanosoma brucei and studies have identified some of the proteins that participate in this HR pathway, the recruitment kinetics of the HR machinery onto DNA during DSB repair have not been clearly elucidated in this organism. Using immunofluorescence, protein DNA-bound assays, and DNA content analysis, we established the recruitment kinetics of the HR pathway in response to the DSBs generated by ionizing radiation (IR) in procyclic forms of T. brucei. These kinetics involved the phosphorylation of histone H2A and the sequential recruitment of the essential HR players Exo1, RPA, and Rad51. The process of DSB repair took approximately 5.5 hours. We found that DSBs led to a decline in the G2/M phase after IR treatment, concomitant with cell cycle arrest in the G1/S phase. This finding suggests that HR repairs DSBs faster than the other possible DSB repair processes that act during the G1/S transition. Taken together, these data suggest that the interplay between DNA damage detection and HR machinery recruitment is finely coordinated, allowing these parasites to repair DNA rapidly after DSBs during the late S/G2 proficient phases.

Transcription activity contributes to the firing of non-constitutive origins in Trypanosoma brucei

“…Such a proposal has previously been supported in this parasite after the induction of DSBs by ionizing radiation (IR), where repair was carried out quickly in late S/G2 within approximately 6 h (39). Thus, we can infer that endogenous DNA damage does not appear to be a great obstacle for this parasite to continue to proliferate, given the efficient DNA repair that most trypanosomatids possess (39,52,59,60).…”

“…The use of this entire pool of origins is necessary to maintain the robustness of S phase duration and seems to be of paramount importance since it also contributes to the maintenance of DNA replication, allowing the survival of this divergent parasite. activated, which help to maintain the robustness of S phase, contributing to the maintenance of 39 DNA replication. Of note, the activation of non-constitutive origins may occur in an active (triggered by DNA replication impairment) or passive (stochastic) manner.…”

“…Alternatively, the extension of stalled forks over longer time periods and the erosion of uncapped telomeres may generate one-ended DSBs, which could also give rise to the formation of endogenous γH2A(X) foci (40,55,56). In general, one-ended DSBs are repaired during S phase by a mechanism similar to homologous recombination (HR) called break-induced replication (BIR) (56), while common two-ended DSBs generated by replication stress are repaired by HR, but in late S and G2 phases (39,40,57,58).…”

“…However, the presence of this variant histone has not been reported in trypanosomatids or yeast (34,37). Several studies indicate that the canonical phosphorylated histone (γH2A) plays the role of γH2AX in these organisms, i.e., increasing in vivo in response to DNA lesions (34,38,39).…”

“…brucei shows endogenous levels of DNA breaks represented by a few γH2A fluorescent foci (red), predominantly throughout the G1, S and G2 phases ( Figure 5A). These foci became rare during the mitosis and cytokinesis phases ( Figure 5A), which can be explained by the fact that, when DNA breaks occur during S phase, their repair takes place predominantly in the late S or G2 phases via the homologous recombination (HR) pathway (39,40). Alternatively, HR repairs oneended DSBs that arise following replication fork collapse throughout S phase (40).…”