Keeping Ku in check: The crucial role of DNA-PKcs

Like beads on a necklace, several Ku protein complexes can thread onto DNA from the ends generated when our DNA is broken. A study published in Cell Reports reveals that the enzyme DNA-PKcs protects our DNA from excessive Ku entry. This work also uncovers two mechanisms that limit Ku accumulation in the absence of DNA-PKcs. The discovery of these mechanisms, which are sometimes deficient in tumor cells, opens up new therapeutic prospects

Many anti-cancer therapies, such as radiotherapy, ensure their efficacy by inducing DNA double-strand breaks, i.e. the rupture of both strands of the DNA molecule, thus generating two ends. In humans, the Ku protein complex binds immediately to each end of the break, thanks to its ring-like structure. This ring can thread and then slide over the DNA, allowing several Ku complexes to load from the end, like pearls on a necklace. Since Ku-coated DNA cannot be opened to allow gene expression, the existence of a mechanism blocking this sliding in cells was suspected for a long time.

A protein holds Ku at the DNA ends
In an article published in Cell Rep., scientists from IPBS used super-resolution fluorescence microscopy to monitor the amount of Ku loading onto X-ray-induced breaks in human cells, mimicking radiotherapy. With this approach, the researchers discovered that the DNA-PKcs protein, which binds directly to Ku at the DNA end, blocks its sliding onto DNA. Although DNA-PKcs is well known for its enzymatic kinase activity, which enables it to modify the properties of several DNA repair proteins, the researchers showed that, in this case, this was a physical role for DNA-PKcs. In collaboration with a team from Harvard University, the researchers showed that this role was conserved in Xenopus, a model organism that researchers used for an in-depth analysis of the composition of repair complexes.

Two mechanisms limit the accumulation of Ku in chromatin
In addition, the researchers have shown that when DNA-PKcs is absent, two mechanisms can limit Ku accumulation. Ku can be modified by grafting with multiple copies of a protein called ubiquitin, a processed called ubiquitination. The resulting poly-ubiquitin chain then serves as a signal for its extraction from DNA. This first mechanism depends on the FBXL12 protein, which recruits a ubiquitin grafting complex. Furthermore, they showed that when breaks are induced during the DNA replication phase, the excess of Ku on the breaks is eliminated by digestion of the DNA by a nuclease complex regulated by the CtIP protein and the ATM kinase.

Under normal conditions, DNA-PKcs associates with Ku at the ends of DNA break and blocks its entry onto DNA. In the absence of DNA-PKcs, Ku accumulates onto DNA, but this accumulation is limited by active elimination dependent on its modification by ubiquitin chains, a process called ubiquitination. When these two systems are absent, Ku accumulates excessively. Ku accumulation at individual breaks can be monitored by fluorescence imaging, by measuring the intensity of Ku foci (SIM images), or by tracking the size of Ku foci using STORM-type super-resolution microscopy.
Copyright: Thumbnail © Sébastien Britton with the help of Midjourney. Illustration : © Sébastien Britton and Madeleine Bossaert.

The excessive amount of Ku blocks the expression of nearby genes
Finally, the researchers studied the consequence of Ku accumulation on gene expression, a process requiring DNA opening, by developing a tool to monitor gene expression near DNA ends. Using this, they confirmed that the accumulation of Ku on DNA in absence of DNA-PKcs blocks the expression of nearby genes.
This work answers a fundamental question that had previously remained unanswered: how is the amount of Ku on each DNA break regulated? It also identifies potential therapeutic targets for killing tumor cells with specific defects in some of these mechanisms.

Reference

Identification of the main barriers to Ku accumulation in chromatin. Madeleine Bossaert, Andrew Moreno, Antonio Peixoto, Marie-Jeanne Pillaire, Pauline Chanut, Philippe Frit, Patrick Calsou*, Joseph John Loparo*, Sébastien Britton*. Cell Rep (2024) https://doi.org/10.1016/j.celrep.2024.114538

Contacts

CNRS Scientist | Sébastien Britton | Sebastien.Britton@ipbs.fr
IPBS press | Françoise Viala | T +33 6 01 26 52 59 | communication@ipbs.fr

Keeping Ku in check: The crucial role of DNA-PKcs