Titolo della tesi: RAD52 prevents accumulation of Polα-dependent replication gaps at perturbed replication forks in human cells
RAD52 (Radiation sensitive 52) is a highly conserved protein involved in DNA damage repair. In humans, RAD52 contributes to the stabilization of the replication forks after replication arrest and prevents an excessive reversion of the forks that may result in pathological strand degradation. Consequently, loss of the RAD52 function leads to excessive replication fork reversal, persistence of under-replicated regions and chromosome instability. However, the mechanisms that the cell uses to overcome the stalled forks and complete the DNA replication in the absence of RAD52 are still unknown. Here, we investigated how replication restarts in absence of a functional RAD52. As recent evidence suggests that an excessive fork reversion could limit the repriming-mediated replication restart, we asked whether the loss of RAD52 could affect this mechanism. Using multiple cell biology experiments, we demonstrated that, in response to hydroxyurea-mediated replication fork slowing or arrest, the impairment of RAD52 ssDNA-binding induces the accumulation of parental DNA gaps due to increased recruitment of DNA polymerase alpha (Polα) and that RAD52 deficiency stimulates Polα-mediated, but not PrimPol-mediated, replication repriming. Furthermore, we showed that Polα recruitment occurs downstream the remodelled reversed fork (RF) and depends on the function of RAD51. Finally, we demonstrated that the repair of ssDNA gaps derived from Polα-mediated repriming is delayed until the G2-phase of the cell cycle, and are filled by the post replicative repair (PRR) mechanism mediated by PCNA ubiquitination. In this scenario, the ssDNA gaps repair is possibly mediated by a post-replicative activation of the checkpoint kinase 1 (CHK1). Collectively, our findings unveil a novel Polα-mediated repriming pathway acting when reversed forks undergo extensive degradation in the absence of fork cleavage. We proposed that the repriming mechanism driven by Polα in absence of RAD52 function is a novel rescue pathway to prevent DNA damage and promote viability under replication stress.