UNRAVELLING THE ROLE OF NOVEL FACTORS INVOLVED IN DOUBLE-STRAND BREAK REPAIR.

Each day every cell of a living organism is constantly exposed to numerous DNA damages deriving both from the environment but also from its own metabolism. The high number of lesions and the consequent genome instability make of DNA damages one of the weightiest challenge to face for a cell. Indeed the ability to detect, recognise and repair a lesion is of pivotal importance, since on these events depend the stability of the genome and, ultimately, cell viability. The main shield eukaryotic cells have evolved to face this challenge is the DNA damage response, a protein network that allow repair of the lesions. Human cells can rely on two main mechanisms to repair double strand breaks, one of the most harmful lesions: homologous recombination and non-homologous end joining. The correct balance between these two pathways depends on cell cycle, chromatin conformation and on the interplay among different factors. In addition, important for the correct pathway choice is the DNA end resection process. It consists in a nucleolytic degradation of the DSB ends to generate a 3' protruding tail to invade the homologous sequence, used as a template to accomplish the HR. Fine regulation of resection is particularly important to correctly repair the damage and prevent genome instability, fuel of cancer. In this Thesis I present the work performed during my three years of PhD, in which I’ve been involved in two projects. Using human cells as a model system I’ve analysed the role of two different proteins, both involved in DNA repair pathway choice: DAXX and SLX4. In the first and half year of my PhD, I analysed the effect of double strand break-dependent phosphorylation of DAXX on its activity as a chaperone of the histone variant H3.3. In brief, we found that upon double strand break, DAXX is phosphorylated by the apical kinase ATM on two serine (S424 S712) and the ability of DAXX to depose H3.3 at the lesion relies on these modifications. The accumulation and maintenance of H3.3 at the damage impact on the histone post-translational modification pattern, impairing 53BP1 protein foci formation and favouring the damage to be repaired through homologous recombination. Our results highlight the important role of histones chaperones and modifications in double strand break repair and suggest a possible mechanism explaining the prediatric glioblastoma occurrence in case of H3.3 mutations. During the last part of my PhD I focused my attention of the role of SLX4 protein in double strand break repair pathway choice. Preliminary data of our laboratory suggested an SLX4 pro-resection activity, favouring homologous recombination occurrence. Staring from these results, I analysed resection in Fanconi Anemia patient-derived cells, SLX4 null. In collaboration with Pablo Huertas’ laboratory (CABIMER, Sevilla, Spain) I verified an impairment of the resection process in these cells, confirming SLX4 pro-resection role. Further analysis will be required to elucidate the molecular mechanism of SLX4 activity but these first results are very promising to shed light on a new player of the intricate network of double strand breaks repair pathway choice.