ATR Kinase Is a Crucial Player Mediating the DNA Damage Response in Trypanosoma brucei

Abstract

DNA double-strand breaks (DSBs) are among the most deleterious lesions that threaten genome integrity. To address DSBs, eukaryotic cells of model organisms have evolved a complex network of cellular pathways that are able to detect DNA damage, activate a checkpoint response to delay cell cycle progression, recruit the proper repair machinery, and resume the cell cycle once the DNA damage is repaired. Cell cycle checkpoints are primarily regulated by the apical kinases ATR and ATM, which are conserved throughout the eukaryotic kingdom. Trypanosoma brucei is a divergent pathogenic protozoan parasite that causes human African trypanosomiasis (HAT), a neglected disease that can be fatal when left untreated. The proper signaling and accuracy of DNA repair is fundamental to T. brucei not only to ensure parasite survival after genotoxic stress but also because DSBs are involved in the process of generating antigenic variations used by this parasite to evade the host immune system. DSBs trigger a strong DNA damage response and efficient repair process in T. brucei, but it is unclear how these processes are coordinated. Here, by knocking down ATR in T. brucei using two different approaches (conditional RNAi and an ATR inhibitor), we show that ATR is required to mediate intra-S and partial G1/S checkpoint responses. ATR is also involved in replication fork stalling, is critical for H2A histone phosphorylation in a small group of cells and is necessary for the recruitment and upregulation of the HR-mediated DNA repair protein RAD51 after ionizing radiation (IR) induces DSBs. In summary, this work shows that apical ATR kinase plays a central role in signal transduction and is critical for orchestrating the DNA damage response in T. brucei.

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

“…Consistent with TbATR having wide roles in T. brucei biology - akin to the emerging data on diverse ATR functions in other eukaryotes - analysis of various cellular phenotypes reveals more pleotrophic effects after its loss than the impaired cell cycle progression, increased DNA damage and altered VSG expression control documented previously 34,35,37 . First, TbATR depleted cells display numerous changes in their nuclear ultrastructure.…”

“…More recently, wider roles for ATR have been recognised, including maintaining the integrity of the nuclear envelope 27,29,30 , nucleolus 28 and centrosome 31 . In the kinetoplastid parasite T. brucei , TbATR acts in cell cycle progression and DNA double-strand break repair 37 , and in mammal-infective BSF cells the kinase acts in monoallelic VSG expression and VSG recombination during host immune evasion by antigenic variation 34 . However, whether such VSG-related functions could explain why loss of TbATR is essential in mammal-infective T. brucei , but not in tsetse-infective cells, is questionable.…”

“…Previous work has clearly implicated ATR in the response of both T. brucei and Leishmania to DNA damage: loss or inhibition of the kinase sensitises the parasites to lesions resulting from alkylation 45 , irradiation 37 and oxidative stress 71 ; in addition, TbATR RNAi in T. brucei results in focal accumulation of RPA and RAD51, and increased levels of yH2A, which have been mapped to the VSG expression sites 34 . Nonetheless, the action of ATR in kinetoplastid DNA replication has not been as clearly described, and its role in preventing the segregation of damaged chromosomes may not be absolute, at least in T. brucei .…”

“…These data indicate wider, essential roles for ATR in African trypanosomes whose basis have not been dissected. Surprisingly, in tsetse-infective (procyclic form, PCF) T. brucei cells RNAi depletion or chemical inhibition of ATR appears not to affect growth, though cell cycle progression and DNA double-strand break repair are impaired 37 . Although many pathways dictating genome repair and cell cycle progression are conserved in T. brucei , these parasites lack identifiable homologs of ATR pathway members, including CHK1 and Cdc25 38 and in the related kinetoplastid parasite Leishmania , divergent 9-1-1 complex functions have been reported 39,40 , suggesting the kinetoplastid ATR pathway may operate distinctly from that of a ‘model’ eukaryote.…”

Unpicking the Roles of DNA Damage Protein Kinases in Trypanosomatids

“…Nonetheless, what aspect of ATR function results in T. brucei death after the loss of the PK is unknown. In this regard, recent work in insect stage T. brucei revealed that depletion of ATR only moderately affects parasite proliferation and cell cycle progression, despite playing an important role during HR and damage signaling in this life cycle stage in response to ionizing radiation (IR) (Marin et al, 2020). This dichotomy likely reflects alternative demands on repair and replication in distinct life cycle stages.…”