Titolo della tesi: ELUCIDATING THE RELEVANCE OF THE UBIQUITIN-BINDING ACTIVITY OF WRNIP1 FOR REPLICATION FORK STABILIZATION AFTER REPLICATION STRESS
Complete and faithful DNA replication is fundamental to cellular proliferation and genome stability. Replication forks frequently encounter obstacles that lead to fork slowdown or stalling termed replication stress. Accurate stabilization of stalled replication forks is of paramount importance to guarantee genome integrity and, in turn, avoid cancer development. A complex network of proteins defends stalled fork stability to avoid its degeneration into dangerous breakage. Among these proteins, we have previously unveiled an important role for the human Werner helicase interacting protein 1 (WRNIP1). It is a member of the AAA+ ATPase family that play a crucial role in safeguarding the integrity of arrested replication forks. Indeed, WRNIP1 is directly involved in preventing pathological MRE11-mediated degradation of stalled forks by promoting RAD51 stabilization at ssDNA in a way that is not dependent from its ATPase activity, which is rather involved in replication fork restart. However, besides its ATPase activity, WRNIP1 also contains an ubiquitin-binding zinc finger (UBZ) activity, whose function is not yet completely uncovered.
In the present study, we examine the relevance of the ubiquitin binding activity of WRNP1 and provide evidence that it plays an essential role in promoting the integrity of replication forks stalled by treatment with agents that cause genome-wide replication stress. Moreover, we establish that, upon replication fork stalling, the UBZ activity of WRNIP1 is strictly required to protect stalled forks from the nucleolytic attack of MRE11, and that it mediates RAD51 stabilization at chromatin. Notably, we prove that the protective function played by the UBZ activity of WRNIP1 relies on the RAD18-dependent PCNA ubiquitination.
Overall, these findings define a previously undescribed role for the UBZ activity of WRNIP1 in safeguarding genome integrity in response to replication stress.