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Radiotherapy: to defend DNA, repair proteins go in search of the Ring

To understand why some cancer cells resist radiotherapy, an international team of researchers used crystallography to capture the first moments of the molecular ballet that allows these cells to repair their DNA. This study was conjointly conducted by the groups of Patrick Calsou (Institut of Pharmacology and Structural Biology, (IPBS) CNRS, University Paul Sabatier - Toulouse III, équipe labellisée Ligue Contre le Cancer) and Jean-Baptiste Charbonnier (Institute of Integrative Biology of the Cell (I2BC), CEA Paris-Saclay, CNRS, University Paris-Sud). It is published in Nature Structural & Molecular Biology.


Radiotherapy is a critical tool in cancer treatment. Prescribed in one in two cases (i.e. 200,000 cases per year in France), the high energy rays used destroy cancer cells by fragmenting their DNA. However, in tumours, some cells can resist treatment by repairing the breaks in their DNA. To increase the effectiveness of radiotherapy, for instance by inhibiting DNA repair within the tumour, researchers first need to get a detailed understanding of how these repair mechanisms work. 
In irradiated cells, a protein complex assembles around a ring-shaped protein known as Ku, which quickly encircles the ends of DNA breaks. The protein ballet ends with the joining of the breaks ends together. 
Researchers studied the first phase of this choreography, with Ku at the centre, and particularly how come on stage APLF and XLF, two proteins that work with Ku. Using X-ray crystallography to view the protein complexes at an atomic scale, they produced snapshots of Ku/APLF and Ku/XLF interactions. For the first time, these images show that each of the two partners comes into contact with Ku on distinct sites. Researchers showed that if these sites are changed, breaks are no longer properly repaired and the cells survive much less after they are irradiated.


Figure 1: Three levels of analysis of the recruitment of repair proteins on DNA breaks (Ku in purple and XLF in green. APLF is not shown for clarity).
From left to right:
1/ accumulation of protein as a spot at the irradiation site in a cell nucleus;
2/ protein aggregates visualized under super-resolution microscopy;
3/ molecular complex characterized by X-ray crystallography: the interaction area between Ku and XLF is shown in yellow; the DNA in brown is surrounded by the Ku ring.
© PCalsou / JBCharbonnie

In the long term, precise knowledge of the contact points between the DNA break repair catalysts could lead to the design of molecules that would perfectly fit to these sites, prevent the assembly of the repair machinery in tumours and potentiate radiotherapy.


Figure 2: Cells containing the XLF protein marked in green (left) are irradiated by laser to create DNA breaks along thin lines. The proteins are recruited in one minute on the parallel lines drawn by the laser (right). © PCalsou / JBCharbonnier


XLF and APLF bind to Ku80 on two remote sites to ensure repair by non-homologous end-joining. (2018) Nemoz C*, Ropars V*, Frit P*, Gontier A, Drevet P, Yu J, Guerois R, Pitois A, Comte A, Delteil C, Barboule N, Legrand P, Baconnais S, Yin Y, Tadi S, Barbet-Massin E, Berger I, Le Cam E, Modesti M, Rothenberg E, Calsou P and Charbonnier JB. Nature Structural and Molecular Biology (*equal contribution) 25(10):971-980

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