Uncoupling of programmed DNA cleavage and repair jeopardizes the assembly of the Paramecium somatic genome

In the ciliate Paramecium, the precise excision of numerous Internal Eliminated Sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double strands breaks (DSBs) are introduced by the PiggyMac endonuclease, and repaired in a highly concerted manner by the Non-Homologous End Joining pathway (NHEJ), as illustrated by the complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show here that expression of a DNA binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but not DSB repair, leading to accumulation of unrepaired DSBs. When wildtype and mutant Ku are co-expressed, the DSBs induced by Ku70-6E can be repaired by wildtype Ku, which uncouples DNA repair from the cleavage step. High-throughput sequencing of the developing MAC genome in these conditions reveals the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. We conclude that coupling the two steps of IES excision ensures that both extremities are maintained together throughout the process, and propose that Ku assists PiggyMac during assembly of the synaptic pre-cleavage complex.