Program > Browse abstracts by author > Iehl Florence

In Paramecium, genome rearrangements involve the precise excision of numerous single-copy relics of mobile elements, called internal eliminated sequences (IESs), from somatic DNA. Rearrangements are initiated by DNA double-strand breaks (DSBs) requiring the endonuclease activity of a domesticated piggyBac transposase, PiggyMac (Pgm), and repaired by the classical non-homologous end joining (NHEJ) pathway. These two steps (DNA cleavage and repair) are tightly linked and RNA interference against KU70 or KU80c (a specialised paralog of KU80) induces a developmental phenotype with complete inhibition of inhibition of DNA cleavage. Using a Ku70 DNA repair defective mutant, we have shown that coupling between DNA cleavage and repair machinery ensures faithful DNA repair of IES flanking sequences. In the absence of coupling, assembly of the new somatic genome leads to numerous errors, including ends repaired by de novo telomeric addition and numerous translocations between IES flanking sequences. We have investigated the role of other components of the NHEJ machinery encoded by the Paramecium genome. All are essential for the survival of the sexual progeny, but their depletion leads to very different molecular phenotypes. Depletion of Xlf or DNAPKcs inhibits DNA repair, whereas the absence of two different homologs of Paxx leads to IES retention, demonstrating that additional NHEJ factors besides Ku70/80 contribute to coupling. The properties of the DNA repair factor required for DNA cleavage and the nature of the errors observed under uncoupling conditions suggest a model for Pgm-dependent cleavage activation. In this model, the Pgm-domesticated transposase is activated by the formation of a synaptic complex based on DNA repair proteins.