Invited Speaker 23rd International Society of Magnetic Resonance Conference 2023

Assembly of DNA repair complexes mediated by intrinsically disordered regions  (#7)

SOPHIE ZINN-JUSTIN 1 , RANIA GHOUIL 1 , MANON JULIEN 1 , ASA EHLEN 2 , CAMILLE GELOT 3 , AURA CARREIRA 4 , RAPHAEL CECCALDI 3 , ROLAND KANAAR 5 , ALEX ZELENSKY 5 , Francois-Xavier Theillet 1 , SIMONA MIRON 1
  1. I2BC-CEA, Gif-sur-Yvette, France
  2. Institut Curie, PSL Research University, CNRS, UMR3348, Orsay, France
  3. Institut Curie, PSL Research University, INSERM U830, Paris, France
  4. Centro de Biologia Molecular Severo Ochoa, CBMSO, CSIC-UAM, Madrid, Spain
  5. Oncode Institute, Erasmus University Medical Center, Rotterdam, Netherlands

To mitigate the threat of DNA double strand breaks (DSBs), human cells rely on the activity of multiple DNA repair machineries, that are tightly regulated throughout the cell cycle. In interphase, DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). The Breast CAncer type 2 protein (BRCA2) is an evolutionarily conserved central regulator of homologous recombination (HR) in most eukaryotes. During HR, BRCA2 delivers the recombinases – RADiation sensitive 51 (RAD51) and Disrupted Meiotic cDNA 1 (DMC1) – at the DNA repair site. Human BRCA2 is large and mostly disordered, it is mutated in several cancers. In mitosis, it was recently discovered that the polymerase Polθ is activated upon phosphorylation to repair DSBs. It mediates joining of broken DNA ends and thereby maintains genome integrity in HR-deficient tumors. It is a new target for drugs against cancers. The central region of Polθ is also large and disordered. Here we will show how, using NMR, we have identified functionally important phosphorylation sites and associated binding events in BRCA2 and Polθ. Monitoring phosphorylation with time in several BRCA2 conserved regions has revealed new docking sites for the kinase PLK1 and the phosphatase PP2A (Ehlen et al., 2020; Alik et al., 2020; Julien et al., 2021). A repeated motif was further identified in BRCA2, which is able to assemble a 1 MDa complex through its binding to the HSF2BP protein (Ghouil et al., 2021 & submitted). Similarly, in Polθ, phosphorylation of two conserved fragments was characterized by NMR, revealing key phospho-sites that regulate the binding of Polθ to the BRCT domains of TopBP1, an event essential for the localization of Polθ to mitotic DSBs (Gelot et al., submitted). The 3D structures of these complexes were described using a combination of NMR, X-ray crystallography and cryo-EM. Their interfaces composed of one or several low affinity contact sites, and the regulation of their assembly by phosphorylation, will be presented and discussed.

  1. Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1. Ehlén Å, Martin C, Miron S, Julien M, Theillet FX, Ropars V, Sessa G, Beaurepere R, Boucherit V, Duchambon P, El Marjou A, Zinn-Justin S, Carreira A. Nat Commun. 2020 Apr 14;11(1):1819. doi: 10.1038/s41467-020-15689-9.
  2. Sensitivity-Enhanced 13 C-NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH. Alik A, Bouguechtouli C, Julien M, Bermel W, Ghouil R, Zinn-Justin S, Theillet FX. Angew Chem Int Ed Engl. 2020 Jun 22;59(26):10411-10415. doi: 10.1002/anie.202002288.
  3. Intrinsic Disorder and Phosphorylation in BRCA2 Facilitate Tight Regulation of Multiple Conserved Binding Events. Julien M, Ghouil R, Petitalot A, Caputo SM, Carreira A, Zinn-Justin S. Biomolecules. 2021 Jul 20;11(7):1060. doi: 10.3390/biom11071060.
  4. BRCA2 binding through a cryptic repeated motif to HSF2BP oligomers does not impact meiotic recombination. Ghouil R, Miron S, Koornneef L, Veerman J, Paul MW, Le Du MH, Sleddens-Linkels E, van Rossum-Fikkert SE, van Loon Y, Felipe-Medina N, Pendas AM, Maas A, Essers J, Legrand P, Baarends WM, Kanaar R, Zinn-Justin S, Zelensky AN. Nat Commun. 2021 Jul 29;12(1):4605. doi: 10.1038/s41467-021-24871-6.
  5. BRCA2-HSF2BP Oligomeric Ring Disassembly by BRME1 Promotes Homologous Recombination. Ghouil R, Miron S, Sato K, Ristic D, van Rossum-Fikkert SE, Legrand P, Ouldali M, Winter JM, Ropars V, David G, Arteni AA, Wyman C, Knipscheer P, Kanaar R, Zelensky A, Zinn-Justin S. in review
  6. Polθ is phosphorylated by Polo-like kinase 1 (PLK1) to enable repair of DNA double strand breaks in mitosis. Gelot C, Kovacs M, Miron S, Mylne E, Boeffard-Dosierre L, Popova T, Dingli F, Loew Damarys, Guirouilh-Barbat J, Del Nery E, Zinn-Justin S, Ceccaldi R. in review