Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia

PLoS Genet. 2014 Aug 28;10(8):e1004552. doi: 10.1371/journal.pgen.1004552. eCollection 2014 Aug.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence / genetics
  • Cell Nucleus / genetics
  • Chromosomes / genetics*
  • DNA Breaks, Double-Stranded*
  • DNA Cleavage
  • DNA Repair
  • DNA, Protozoan / genetics
  • Gene Rearrangement / genetics*
  • Genome
  • Genomic Instability*
  • Germ Cells
  • Paramecium tetraurelia / genetics*
  • Transposases / metabolism

Substances

  • DNA, Protozoan
  • Transposases

Grants and funding

This work was supported by core funding from the Centre National de la Recherche Scientifique (CNRS, http://www.cnrs.fr/), and by grants from the Agence Nationale de la Recherche (ANR 2010-BLAN-1603 and ANR-12-BSV6-0017, http://www.agence-nationale-recherche.fr/) and the Fondation ARC (#SFI20121205487, http://www.fondation-arc.org/). AM was supported by PhD fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche (http://www.enseignementsup-recherche.gouv.fr/) and from the Fondation ARC (http://www.fondation-arc.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.