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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.
Highlights
DNA double-strand breaks (DSBs) are among the most toxic forms of DNA damage that threaten genomic integrity
The second chloro2 -deoxyuridine (CldU) pulse labeling was used for three purposes: (i) to identify cells that were replicating in the first pulse and were still replicating, (ii) to identify cells that were not replicating during the first pulse but entered the S phase at the established timepoints during the second pulse, and (iii) to identify cells that were in the S phase during the first pulse but exited the S phase during the second pulse
These results suggest that procyclic T. brucei ATR acts as an apical kinase to coordinate the DNA damage response to IRinduced DSBs
Summary
DNA double-strand breaks (DSBs) are among the most toxic forms of DNA damage that threaten genomic integrity. It can be induced via the effect of cellular metabolites or by DNA-damaging agents (e.g., ionizing radiation) (van Gent et al, 2001). The ATR in Trypanosoma brucei DNA Damage eukaryotic cells of model organisms have a complex evolutionarily conserved network of cellular pathways known as the DNA damage response (DDR) that orchestrates the detection and repair of a wide range of DNA damage (Zhou and Elledge, 2000; Harper and Elledge, 2007; Jackson and Bartek, 2009).
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