Deciphering the interstrand crosslink DNA repair network expressed by <i>Trypanosoma brucei</i>

Abstract Interstrand crosslinks (ICLs) represent a highly toxic form of DNA damage that can block essential biological processes including DNA replication and transcription. To combat their deleterious effects all eukaryotes have developed cell cycle-dependent repair strategies that coopt various factors from ‘classical’ DNA repair pathways to resolve such lesions. Here, we report that Trypanosoma brucei , the causative agent of African trypanosomiasis, possesses such systems that show some intriguing differences to those mechanisms expressed in other organisms. Following the identification of trypanosomal homologues encoding for CSB, EXO1, SNM1, MRE11, RAD51 and BRCA2, gene deletion coupled with phenotypic studies demonstrated that all the above factors contribute to this pathogen’s ICL REPAIRtoire with their activities split across two epistatic groups. We show that one network, which encompasses TbCSB, TbEXO1 and TbSNM1, may operate throughout the cell cycle to repair ICLs encountered by transcriptional detection mechanisms while the other relies on homologous recombination enzymes that together may resolve lesions responsible for the stalling of DNA replication forks. By unravelling and comparing the T. brucei ICL REPAIRtoire to those systems found in its host, targets amenable to inhibitor design may be identified and could be used alongside trypanocidal ICL-inducing agents to exacerbate their effects. Author summary Parasites belonging to the Trypanosoma brucei complex cause a human and animal infections collectively known as African trypanosomiasis. Drugs used against these diseases are problematic as medical supervision is required for administration, they are costly, have limited efficacy, may cause unwanted side effects while drug resistance is emerging. Against this backdrop, there is a need for new therapies targeting these neglected tropical diseases. Previous studies have shown compounds that induce DNA interstrand crosslinks (ICLs) formation are effective trypanocidal agents with the most potent invariably functioning as prodrugs. Despite the potential of ICL-inducing compounds to treat African trypanosomiasis little is known about the ICL repair mechanisms expressed by trypanosomes. Using a combination of gene deletion and epistatic analysis we report the first systematic dissection of how ICL repair might operate in T. brucei , a diverged eukaryote. It sheds light on the conservation and divergence of ICL repair in one of only a handful of protists that can be studied genetically, and offers the promise of developing or exploiting ICL-causing agents as new anti-parasite therapies. These findings emphasise the novelty and importance of understanding ICL repair in T. brucei and, more widely, in non-model eukaryotes.