Cooperation between transposons and DNA repair in their hosts

• Applicant: Dr. Orsolya Barabas
• Subject Area: Biochemistry ; General Genetics and Functional Genome Biology ; Bioinformatics and Theoretical Biology ; Structural Biology ; Cell Biology
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 397026574

Two antagonistic forces control genome evolution. DNA repair pathways safeguard genome stability against genotoxic stress, while mobile genetic elements keep changing genomic sequence and expression profile, ensuring genome plasticity and driving evolution and adaptation.Transposable elements (TEs) are often viewed as parasitic elements, which move from one locus to another in their host genome. Most TEs move by a cut-and-paste process and excise from their donor site before integrating into a new locus. Cut-and-paste TEs usually encode a transposase (Tnpase) with three acidic catalytic residues (DD(D/E)) that cleave DNA at TE ends. This cleavage creates a chromosomal double-strand break (DSB) at the excision site, posing a threat for host genome integrity that can lead to cell death. Strikingly, despite their potentially deleterious effects, some cut-and-paste Tnpases have been domesticated during evolution to carry out essential programmed genome rearrangements, making efficient DSB repair even more critical for their hosts.This project will investigate the extent and molecular mechanisms through which DD(D/E) Tnpases - including domesticated ones - interact with host DNA repair machineries to control the balance between genome stability and plasticity. We will focus on two prominent systems: (i) the Sleeping Beauty Tnpase (SB), frequently used for transgenesis and genetic screens in vertebrates and (ii) PiggyMac (Pgm), a 'domesticated' piggyBac-like Tnpase that conducts programmed elimination of TEs and transposon-related internal eliminated sequences (IES) from the somatic genome of the ciliate Paramecium. Published evidence for an interaction of several Tnpases - including SB and Pgm - with the DSB repair initiator host proteins, Ku70, Ku80 and/or DNA-PKcs, has led to the hypothesis that Tnpases actively recruit non-homologous end-joining (NHEJ) factors to facilitate repair of the TE excision sites. In Paramecium, Ku proteins are even required to 'license' Pgm for DNA cleavage, suggesting that the Pgm/Kuinteraction has acquired an additional function ensuring an intimate coupling of DNA cleavage with DSB repair for the domesticated Tnpase.We propose a multidisciplinary work plan to (i) characterize Tnpase/Ku interactions using co-purification, fluorescent detection of interactions in vivo, and structural biology; (ii) investigate the functional impact of the interactions through selection or design of interaction mutants; (iii) evaluate conservation of the interaction for other DD(D/E) Tnpases and Ku proteins via experimental and bioinformatic approaches. By providing insight into the coordination of transposition and DNA repair our results may also help develop safer transposition tools for biotechnological and medical applications.

• DFG Programme: Research Grants
• International Connection: France
• Cooperation Partner: Dr. Mireille Bétermier