title: Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.
abstract: During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage factory, enabling tight coupling between DSB introduction and repair during PGR.
authors.affiliation: CNRS UPR3404 Centre de Génétique Moléculaire, Gif-sur-Yvette, France; Université Paris-Sud, Département de Biologie, Orsay, France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Marmignon
authors.affiliation: CNRS UPR3404 Centre de Génétique Moléculaire, Gif-sur-Yvette, France; Université Paris-Sud, Département de Biologie, Orsay, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Bischerour
authors.affiliation: CNRS UPR3404 Centre de Génétique Moléculaire, Gif-sur-Yvette, France; Université Paris-Sud, Département de Biologie, Orsay, France.
authors.forename: Aude
authors.initials: A
authors.lastname: Silve
authors.affiliation: CNRS UPR3404 Centre de Génétique Moléculaire, Gif-sur-Yvette, France; Université Paris-Sud, Département de Biologie, Orsay, France.
authors.forename: Clémentine
authors.initials: C
authors.lastname: Fojcik
authors.affiliation: CNRS UPR3404 Centre de Génétique Moléculaire, Gif-sur-Yvette, France; Université Paris-Sud, Département de Biologie, Orsay, France.
authors.forename: Emeline
authors.initials: E
authors.lastname: Dubois
authors.affiliation: CNRS UPR3404 Centre de Génétique Moléculaire, Gif-sur-Yvette, France; Université Paris-Sud, Département de Biologie, Orsay, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Arnaiz
authors.affiliation: CNRS UPR3404 Centre de Génétique Moléculaire, Gif-sur-Yvette, France; Université Paris-Sud, Département de Biologie, Orsay, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Kapusta
authors.affiliation: Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, Paris, France; INSERM, U1024, Paris, France; CNRS, UMR 8197, Paris, France; Université Paris Diderot, Sorbonne Paris Cité, UFR Sciences du Vivant, Paris, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Malinsky
authors.affiliation: CNRS UPR3404 Centre de Génétique Moléculaire, Gif-sur-Yvette, France; Université Paris-Sud, Département de Biologie, Orsay, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Bétermier
mesh_terms: D001483:Base Sequence / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics; D002875:Chromosomes / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D053837:DNA Cleavage; D004260:DNA Repair; D016054:DNA, Protozoan / Q000235:genetics; D015321:Gene Rearrangement / Q000235:genetics*; D016678:Genome; D042822:Genomic Instability; D005854:Germ Cells; D016805:Paramecium tetraurelia / Q000235:genetics*; D019895:Transposases / Q000378:metabolism
chemical_list: D016054:DNA, Protozoan; D019895:Transposases