Dissecting the Structure and Interactions of the MRN Complex in Trypanosoma brucei Genome Maintenance

Descrizione del progetto

Meccanismi di riparazione del DNA nei tripanosomi

La tripanosomiasi africana, comunemente nota come malattia del sonno, è una malattia parassitaria trasmessa dalla mosca tse-tse. Causata dal parassita Trypanosoma brucei, colpisce sia gli esseri umani che gli animali in tutta l’Africa subsahariana. Il parassita elude il sistema immunitario modificando continuamente le proteine presenti sulla sua superficie, un processo legato ai meccanismi di riparazione del DNA. Con il sostegno del programma di azioni Marie Skłodowska-Curie, il progetto MIDAS si propone di studiare un complesso fondamentale di riparazione del DNA presente in T. brucei, in grado di riparare le rotture a doppio filamento del DNA e di contribuire all’evasione immunitaria. L’obiettivo è quello di determinare la struttura e la rete di interazioni di questo complesso e di mettere in luce i meccanismi specifici del parassita. I risultati del progetto potrebbero fornire nuove importanti informazioni su come i tripanosomi africani riescano a eludere il sistema immunitario dell’ospite.

Obiettivo

Double-strand breaks (DSBs) are among the most dangerous DNA lesions, and their repair is essential for genome stability. Central to this process is the MRN complex (MRE11–RAD50–NBS1), which senses breaks, processes DNA ends, activates ATM, and coordinates pathway choice between homologous recombination (HR), non-homologous end joining (NHEJ), and alternative end-joining (MMEJ). At chromosomal breaks MRN promotes resection and repair, while at telomeres its activity is restrained to avoid inappropriate repair. In Trypanosoma brucei, the parasite responsible for human and animal African trypanosomiasis, DSB repair is crucial not only for genome integrity but also for antigenic variation. This process enables immune evasion by switching expression of variant surface glycoprotein (VSG) genes at subtelomeres. While MRN is required for HR at intrachromosomal breaks and restrains recombination at VSG sites, its structural features, regulation, and interaction partners in T. brucei remain unexplored. No high-resolution structure exists, and its divergence limits computational predictions. This project will elucidate how the T. brucei MRN complex regulates DSB repair in different genomic contexts, intrachromosomal and subtelomeric loci. We will (i) determine its structure by cryo-EM with ATP and DNA, defining domain organization, DNA/nucleotide contacts, and nuclease regulation; (ii) validate structural determinants through mutagenesis of RAD50–MRE11 and nuclease interfaces, assessing stability, ATPase, and nuclease activity; and (iii) identify MRN interactors in vivo using proximity labeling at intrachromosomal and subtelomeric DSBs. By combining structural and functional approaches, this work will reveal conserved and parasite-specific mechanisms of MRN regulation, clarify its role in genome stability and antigenic variation, and provide insights into DNA repair in a divergent eukaryote of medical importance.