Cooperation between transposons and DNA repair in their hosts
General Genetics and Functional Genome Biology
Bioinformatics and Theoretical Biology
Structural Biology
Cell Biology
Final Report Abstract
Two antagonistic forces shape genome evolution. DNA repair pathways safeguard genome stability against harmful modifications, while mobile genetic elements continuously alter genomic sequence and expression profile, facilitating genome plasticity, evolution and adaptation. Transposable elements (TEs) are commonly viewed as parasitic DNA that can relocate within their host genome. Most TEs move by a cut-and-paste process, excising themselves from their donor site before integrating into a new location. TE movement is catalyzed by a transposase (Tnpase) protein that cleaves 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 potential harmful effects, certain 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 host organisms. This project investigated the extent and molecular mechanisms through which Tnpases interact with host DNA repair machineries to control the balance between genome stability and plasticity. We focused on two prominent systems: (i) the Sleeping Beauty Tnpase (SB), a widely used genetic engineering tool in vertebrates and (ii) PiggyMac (Pgm), a “domesticated” piggyBac-like Tnpase that conducts developmentally programmed DNA rearrangements in the ciliate Paramecium. Previous evidence indicated that the SB and Pgm Tnpases interact with the DSB repair initiator proteins, Ku70/Ku80, actively recruiting the non-homologous end-joining (NHEJ) machinery to facilitate the repair of TE excision sites. Using a multidisciplinary work plan we aimed to (i) characterize Tnpase/Ku interactions by biochemistry, cell biology and structural biology methods; (ii) investigate the impact of the interactions via mutagenesis and functional assays; (iii) explore the conservation of the interaction by evolutionary analysis of diverse Tnpases. Due to the unforeseen circumstances of the COVID-19 pandemic and technical challenges, certain project objectives were delayed or impaired. Nevertheless, we achieved profound and groundbreaking results. For Pgm, recombinant protein reconstitution and structural studies proved to be exceptionally challenging. However, thanks to revolutionary advances in artificial intelligence technologies, we succeeded to construct a structural model for the Pgm protein and use it to predict interactions with the DNA repair machinery. Additionally, we resolved the crystal structure of the PtKu70a/PtKu80c complex, which revealed key differences to the human homologues and visualized its so far ambiguous C-terminal protein interaction domain. This data provides valuable insights into species-specific differences in DNA repair and its molecular cooperation with transposition. Importantly, we further succeeded to determine the first structure of a complete SB Tnpase - DNA complex, a milestone that had eluded researchers for decades. The structure revealed an intertwined interaction between SB and transposon DNA and explained how specific intermolecular contacts and biochemical mechanisms make SB highly efficient in transposition. This data will help further investigate how SB communicates with human DNA repair proteins and provides an invaluable resource for the design of advanced SB Tnpase variants to develop safer genome engineering tools for biotechnological and medical applications. Overall, despite the challenges faced, our project achieved significant milestones and yielded landmark results in the mechanistic analysis of Tnpases and their cooperation with the DNA repair machinery of their host cells. The resulting insights will greatly propel research in the field and stimulate future development of a new generation of genome engineering technologies.
Publications
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Jump ahead with a twist: DNA acrobatics drive transposition forward. Current Opinion in Structural Biology, 59, 168-177.
Arinkin, Vladimir; Smyshlyaev, Georgy & Barabas, Orsolya
