Cells are continuously exposed to DNA damaging agents that cause different types of lesions. Double strand breaks (DSBs), where both strands of DNA are broken, are the most toxic lesions. To repair DSBs and ensure genome stability and cell survival, mammalian cells evolved two main pathways, Homologous recombination (HR) and classical Nonhomologous end joining (c-NHEJ). Failure in these pathways triggers genome instability, which correlates with tumorigenesis and cancer progression, but can also contribute to cancer treatment when properly exploited. At the same time, the end of our chromosomes, the telomeres, should be protected from being sensed as DSBs and aberrantly repaired. In fact, failure in telomere protection due to short or dysfunctional telomeres can cause chromosome fusions, genome instability and, in some cases, contribute to tumorigenesis. To this end, telomeres are protected by the shelterin complex in all cell types and elongated by telomerase in germline and somatic stem cells. 

This thesis aims to find new factors involved in the DNA damage response (DDR) that could be used as markers for cancer diagnosis and/or targets in cancer therapy. 

In paper I, we explore the effect of two novel variants in the H/ACA RNA binding complex component NHP2 identified in a patient with Aplastic anemia, gastric cancer, and signs of premature aging. H/ACA RNA binding complex is essential for the stability and maturation of both ribosomal RNA and the telomerase RNA component hTR. By in silico and in cells analysis, we found that both mutations reduce the affinity of NHP2 for the other components of the H/ACA RNA binding complex due to the misplacement of the N-terminus, affecting protein stability. Furthermore, these variants cause reduced telomerase activity by failing to preserve hTR. However, they do not affect the DDR. 

In papers II and III, we investigated the role of chromatin mobility in the maintenance of genome stability. In fact, the mobility of DSBs can cause translocations, one of the main hallmarks of cancer initiation and progression, but is also one of the mechanisms responsible for the efficacy of therapies based on PARP inhibition against breast and ovarian cancer defective in HR. In paper II, we describe a method for consistent and unbiased quantification of DSB mobility and nuclear deformations in the presence of multiple DSBs by fluorescent live-cell imaging. This method can be used with any fluorescent-tagged proteins binding specific genome regions, such as telomeres. In paper III Section A, we analyzed DSB mobility and mis-repair in the context of nuclear envelope (NE) alterations as Lamin A/C deletion or reduction in sphingolipids synthesis. In fact, previous studies have shown that different components of the NE are involved in promoting and/or counteracting such mobility, but the role of the NE itself was not yet explored. By combining genetic modifications and/or chemical inhibition with microscopy techniques, we found that the presence of invaginations significantly increases DSB mobility and mis-repair after treatment with PARP inhibitor in the absence of BRCA1, suggesting that the visualization of the NE could be used as marker for predicting the cancer therapy outcome and that, in the long run, the NE structure could be used as target in cancer therapy. In paper III Section B, we demonstrated that cells subjected to mechanical shear stress and/or to an agonist of the mechanosensor channel Piezo1 reduced DSB mobility and mis-repair. This effect is associated with chromatin decompaction, as observed by electron microscopy and ATAC sequencing, and reduced telomere mobility, suggesting that the shear stress-mediated activation of Piezo1 promotes the chromatin opening and that chromatin compaction can influence DSB mobility and repair. Since cancer cells are exposed to mechanical stress during metastasis, our work suggests that cotreatment with drugs closing the chromatin and/or inhibiting Piezo1 could increase the sensitivity of cancer cells to chemotherapy.