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.
Highlights
DNA double strand breaks (DSBs) are among the most deleterious DNA lesions: if left unrepaired, a single double-strand breaks (DSB) may trigger cell death, while incorrect repair can give rise to chromosome rearrangements [1]
DNA double-strand breaks (DSBs) are potential threats for chromosome stability, but they are usually repaired by two major pathways, homologous recombination or nonhomologous end joining (NHEJ)
We use the ciliate Paramecium tetraurelia as a unicellular model to study how DNA breakage and DSB repair are coordinated during programmed genome rearrangements
Summary
DNA double strand breaks (DSBs) are among the most deleterious DNA lesions: if left unrepaired, a single DSB may trigger cell death, while incorrect repair can give rise to chromosome rearrangements [1]. Cells rely on two major pathways to repair DSBs. Homologous recombination (HR) uses a homologous template to restore the sequence of the broken chromosome, while non-homologous end joining (NHEJ) proceeds through the ligation of free DNA ends. Homologous recombination (HR) uses a homologous template to restore the sequence of the broken chromosome, while non-homologous end joining (NHEJ) proceeds through the ligation of free DNA ends Even though they can be very toxic, programmed DSBs are obligatory intermediates in essential biological processes, such as meiosis or acquired immune response. The Spo endonuclease cleaves DNA and DSB repair is carried out by HR [2]. During lymphocyte differentiation, programmed genome rearrangements (PGR) mediated through V(D)J recombination generate the large diversity of immunoglobulin genes [3].
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