CE11 - Caractérisation des structures et relations structure-fonctions des macromolécules biologiques

Deciphering the protein choreography orchestrated by Ku threaded onto broken DNA – BreakDance

Submission summary

Double-strand breaks (DSBs), despite being deleterious DNA lesions, are intentionally generated during radiotherapy or in genome editing by CRISPR-Cas9. The classical Non-Homologous End Joining (c-NHEJ) is the main DSB repair pathway in human. Understanding the molecular basis of the c-NHEJ is central to improve these major applications. The Ku70/Ku80 (Ku) heterodimer recognizes the DSBs ends, iteratively recruits the NHEJ ligase, nucleases and polymerases in part through their Ku Binding Motifs (KBMs). Ku also contributes to the tethering (synapse) between the two DSB ends to avoid misrepair with other DSB ends. This protein choreography around Ku is still poorly understood. Understanding the assembly and disassembly of the c-NHEJ factors containing a KBM motif on the Ku/DSB ends and the way they contribute to tether the two DSB ends is the main objective of this proposal.

The BreakDance project is focused on 6 c-NHEJ factors that contain one or several KBMs necessary for their recruitment by Ku at DSB ends. These factors are the ligation factors, XLF and PAXX, the nucleases APLF and WRN and the two Pol X polymerases, Pol mu and TdT. Partners 1, 2 and 4 reported the first crystal structures of a Ku/DNA complex with KBMs peptides (Nemoz, 2018, NSMB). We characterized two remote binding sites for APLF and XLF KBMs on the Ku80 subunit, showed an unexpected opening of Ku80 and identified in cells a cooperative role of APLF and XLF in the c-NHEJ. Previously, Partners 1 and 4 studied the KBM of PAXX and showed that it interacts with the Ku70 subunit (Tadi, 2016, Cell Reports).

A first major objective of the project will be to extend the successful strategy used for APLF and XLF to characterize the structure and function of the interaction between Ku/DNA complex and 6 other KBMs. We will thus combine structural, biochemical and cellular analyses on the KBM of PAXX, of WRN (that has 3 KBMs), and of Pol mu and TdT. This first task will provide a complete view on the interaction sites of the KBMs on Ku and will unveil the synergies and competitions in space and in time between most KBM-containing c-NHEJ factors.

A second objective will be to characterize the position of full-length proteins with KBM motifs relatively to the Ku/DNA complex to evaluate how the KBM contribute to orient the functional sites of the proteins relatively to the DSB ends. We will focus on PAXX and on WRN domains bound to Ku/DNA complexes, as Partners 1 and 3 already obtained first 2D cryoEM classes for these complexes.

A third objective is to define the molecular bases of the tethering of the two Ku/DSB ends complex in presence of the ligation complex LIG4/XRCC4/XLF complex or of the PolX polymerases. All partners will combine their expertise to characterize these two major tethering events of the c-NHEJ.

The BreakDance proposal includes several objectives and sub-objectives with increasing levels of difficulties that are independent and feasible in parallel. All objectives will benefit from strong preliminary data obtained by all partners in collaboration. The consortium has all expertise to succeed in this challenging project. We will develop new original tools that will be useful in the genome integrity field: (i) state-of-the-art protein production technologies in several hosts; (ii) integrated approach in structural biology (Xray crystallography, cryoEM & SAXS) ; (iii) a large ensemble of biochemical and biophysical assays for DNA binding, ligation, polymerase and tethering activities; (iv) robust cellular assays to measure recruitments at DSB ends, repair activity of mutants.

The expected outcomes of the proposal are a complete structural, biochemical and cellular description of the molecular mechanisms of assembly and disassembly of main c-NHEJ factors on Ku/DSB ends. The project will set the stage for the targeted inhibition of c-NHEJ that in the future will potentially enhance the efficacy of radiotherapy and genome editing procedures.

Project coordination

Jean-Baptiste CHARBONNIER (Institut de Biologie Intégrative de la Cellule)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

I2BC Institut de Biologie Intégrative de la Cellule
IPBS INSTITUT de PHARMACOLOGIE et de BIOLOGIE STRUCTURALE
CRCM Centre de recherche en cancérologie de Marseille
IP - DSMB Institut Pasteur - Unité de Dynamique structurale des macromolécules biologiques

Help of the ANR 609,811 euros
Beginning and duration of the scientific project: December 2020 - 48 Months

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