SLX4/FANCP controls checkpoint signalling and DNA resection at double-strand breaks

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Using Saccharomyces cerevisiae as a model system, we recently showed that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5\u2032 strand. We are currently investigating SLX4/FANCP role in DSB resection and checkpoint signalling in human cells. Our preliminary results suggest that SLX4 silencing leads to slower DSB resection in U2OS cell line stably expressing the inducible restriction enzyme AsiSI. Moreover, SLX4 may play a role in adaptation to the DNA damage checkpoint arrest in human cells, similarly to our results obtained in yeast. In fact, following ionizing radiation\u2013induced G2 checkpoint, a small percentage of U2OS cells enter mitosis (monitored as appearance of phosphorylated histone H3 foci) with \u3b3-H2AX foci, a marker for unrepaired DNA double-strand breaks. Exit from the G2 checkpoint is delayed depleting SLX4. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity. Since mutations in human SLX4 lead to Fanconi anemia, a genetic disorder associated with high checkpoint marker activation, which could be a cause of bone marrow failure, our results might get new insight into the pathogenesis of the disease