Adjusting for better sealing: the function of DNA polymerases in repairing DNA double-strand breaks
The most serious damage to the DNA molecule is the simultaneous breaking of its two component strands. An international collaboration has just revealed in detail how DNA-copying enzymes are involved in repairing this type of DNA damage in human cells. Ultimately, intimate knowledge of the players involved in this mechanism could enable it to be manipulated to make cancer cells more sensitive to therapies whose mode of action is to break DNA. Published on May 6, 2025, in Nature Communications, this study presents significant advancements in the field.
The DNA that carries our genetic heritage is constantly being damaged by molecules or radiations in the environment. The most serious damage is the simultaneous breaking of the two DNA strands. This damage, known as a DNA double-strand break, can be fatal when a cell is unable to repair it. However, human cells are equipped with a highly efficient repair pathway, the non-homologous end joining (NHEJ), which seals the ends of the break together. The NHEJ can even clean up damaged ends to make a perfect fit before they are welded together. Among the enzymes required, it was known that enzymes that copy DNA, called DNA polymerases, were involved in the adjustment stage, but how they did this was still unclear.
An international collaboration involving Sébastien Britton’s team at the IPBS (CNRS/University of Toulouse) has just revealed in detail how these enzymes participate in the repair of DNA double-strand breaks by NHEJ. In human cells, the Ku protein complex binds immediately to each end of the break thanks to its ring structure. In recent years, various assemblies of several proteins that orchestrate the NHEJ mechanism have been characterised, thanks in particular to advances in the techniques used to study their structure (crystallography, cryo-electron microscopy – cryo-EM). Remarkably, Ku is the common anchor point for these different complexes.
Figure legend: The Ku70/80 ring (orange/green) around the DNA (yellow) is an anchor point for the binding of Pol λ (BRCT domain, purple), Ligase IV (BRCT1 domain, red), PAXX (P-KBM domain, cyan), XLF (X-KBM domain, pink) and APLF (A-KBM domain, brown), proteins involved in the repair of DNA double-strand breaks by NHEJ.
This new study adds a new stone to the edifice by presenting several structures of DNA polymerases interacting with Ku on a DNA break. These structures reveal a specific interaction site between Ku and a domain called BRCT, which is common to DNA polymerases X, the family involved in NHEJ. To prove that this interaction occurs in cells, the contact surface between these proteins was changed by mutations and the consequences of these changes were measured on the cells’ ability to repair DNA breaks. Among the techniques used, the researchers constructed an original tool that measures DNA copying activity on the ends of a DNA molecule broken at a precise point and introduced into cells. The results demonstrate the necessity of the BRCT domain of DNA polymerases X for their recruitment and activity within the NHEJ complex and ultimately for the survival of cells in the presence of a double breaks in their DNA.
Radiotherapy and other anti-cancer therapies act by cutting both strands of the tumour cells DNA, thus generating a toxic excess of DNA double-strand breaks. Ultimately, intimate knowledge of all the players involved in the NHEJ mechanism could enable its modulation to make cancer cells more sensitive to these therapies.
Reference
Structural and Functional Insights into the Interaction between Ku70/80 and Pol X Family Polymerases in NHEJ.
Philippe Frit†, Himani Amin†, Sayma Zahid†, Nadia Barboule†, Chloe Hall, Gurdip Matharu, Steven W. Hardwick, Jeanne Chauvat, Sébastien Britton, Dima Y. Chirgadze, Virginie Ropars, Jean-Baptiste Charbonnier, Patrick Calsou*, Amanda K. Chaplin*. Nat Commun 16(1):4208 †equal contribution, * corresponding authors
Contact
Patrick Calsou (Inserm) or Sébastien Britton (CNRS) | calsou@ipbs.fr | sebastien.britton@ipbs.fr