Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining
A. Model for initial cleavage at IES boundaries by the PiggyMac-associated complex. Nicking of the first strand would liberate a reactive 3′OH residue at IES ends. The putative nucleophilic attack of the top strand is represented by arrows with question marks. B. DNA intermediates and end joining factors involved in IES excision are shown. In wild-type conditions, repair of the chromosomal excision sites (left) is probably mediated by alignment of 4-base 5′ overhangs via the pairing of their central conserved TA. The formation of IES circles (right) may require the resolution of putative hairpins at IES ends, prior to the formation of intramolecular paired-end intermediates. The actors involved in the controlled processing of ends are still unknown. At least for the MAC chromosome junctions, the removal of the 5′ terminal nucleotide does not require Lig4p/Xrcc4p, in contrast to the gap filling step, which is strongly inhibited in LIG4 knock-downs. The final ligation is totally Lig4p/Xrcc4p-dependent. In LIG4 or XRCC4 silencing, no MAC junctions are formed and DSBs accumulate. No circle junctions could be detected.