Identification of the main barriers to Ku accumulation in chromatin
Madeleine Bossaert,
Andrew T. Moreno,
Antonio Peixoto,
Marie-Jeanne Pillaire,
Pauline Chanut,
Philippe Frit,
Patrick Calsou,
Joseph J. Loparo,
Sébastien Britton
Affiliations
Madeleine Bossaert
Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III – Paul Sabatier (UT3), Toulouse, France; Equipe Labéllisée la Ligue contre le Cancer 2018
Andrew T. Moreno
Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
Antonio Peixoto
Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III – Paul Sabatier (UT3), Toulouse, France; Equipe Labéllisée la Ligue contre le Cancer 2018
Marie-Jeanne Pillaire
Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III – Paul Sabatier (UT3), Toulouse, France; Equipe Labéllisée la Ligue contre le Cancer 2018
Pauline Chanut
Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III – Paul Sabatier (UT3), Toulouse, France; Equipe Labéllisée la Ligue contre le Cancer 2018
Philippe Frit
Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III – Paul Sabatier (UT3), Toulouse, France; Equipe Labéllisée la Ligue contre le Cancer 2018
Patrick Calsou
Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III – Paul Sabatier (UT3), Toulouse, France; Equipe Labéllisée la Ligue contre le Cancer 2018; Corresponding author
Joseph J. Loparo
Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Corresponding author
Sébastien Britton
Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III – Paul Sabatier (UT3), Toulouse, France; Equipe Labéllisée la Ligue contre le Cancer 2018; Corresponding author
Summary: Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1–2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.
