Dottorato in BIOLOGIA UMANA E GENETICA MEDICA

Titolo della tesi: Contribution of R-loopaccumulation to genomicinstability in cells with defective replication checkpoint

Conflicts between replication and transcription areacommon source of genomic instability, a characteristic of almost all human cancers. One of the major obstacles to the replication fork progression is transcription. The main transcription-associated structures that can be detrimental to fork movement are R-loops. Notably, a growing number of factors is involved in the resolution of these harmful structures and many are perhaps still unknown. In the first part of this thesis,we implicate the Werner syndrome protein (WRN) in R-loop metabolism. WRN is a well-known fork-protection factor that belongs to the RecQ family of DNA helicases that,when lost,causes the Werner syndrome (WS), a human disorder associated with chromosomal instability and cancer predisposition. WRN plays its major function in genome integrity maintenance, by promoting replicationfork stability after various formsof replication stress. Under mild replication stress, WS cells show impaired ATR-mediated CHK1 activation. However, it remains unclear if WS cells elicit another repair pathway. Here, we demonstrate that loss of WRN leads toenhanced ATM phosphorylation upon mild replication stress, as that induced by prolonged exposure to aphidicolin, resulting in CHK1 activation. Moreover, we find that loss of 9WRN sensitises cells to replication-transcription collisions and promotes accumulation of R-loops, which undergo XPG-dependent cleavage responsible for ATM signalling activation. Importantly, we observe that ATM pathway limits chromosomal instability in WS cells. Finally, we prove that, in WS cells, genomic instability enhanced upon chemical inhibition of ATM kinase activity is counteracted by direct or indirect suppression of R-loop formation or by XPG abrogation (Marabitti et al, 2019).In the second part of this thesis, we explore the role of the Werner interacting protein 1 (WRNIP1)-mediated response in limiting R-loop-associated genomic instability upon mild replication stress in WS cells and, more in general, in cells with compromised ATR-mediated checkpoint response.Using human cellular models withcompromised ATR-dependent checkpoint activation, we show that WRNIP1 is stabilised in chromatin and is needed tomaintain genome integrity by mediating the ATM-dependent phosphorylation of CHK1. Furthermore, we demonstrate that loss of WRN or ATR signalling leads to formation of R-loop-dependent ssDNA upon mild replication stress, which is covered by RAD51. We provide evidencethat WRNIP1 chromatin retention is also required to stabilise the association of RAD51 with ssDNA in proximity of R-loops. Therefore, in these pathological contexts, ATM 10inhibition or WRNIP1 abrogation is accompanied by increased levels of genomic instability. Therefore, these resultssuggesta novel function for WRNIP1 in preventing R-loop-driven genome instability(Marabitti et al, 2020).Overall, these findings allow us to proposea potential role of the WRNIP1-mediated response as regulator of R-loop-associated genomic instability in WS cells and, more in general, in cells with dysfunctional ATR checkpoint, providing new clues to understand the way replication-transcription conflicts are handled, and also strengthening the notion that conflicts between replication and transcription can affect DNA replication, leading to human disease and cancer..