Biology subjects

Valenzisi, P.

Publications and source records attributed to Valenzisi, P..

4 recordsLinked to original sources

bioRxiv2023

PHOSPHORYLATION-DEPENDENT ASSOCIATION OF WRN WITH RPA IS REQUIRED FOR RECOVERY OF REPLICATION FORKS STALLED AT SECONDARY DNA STRUCTURES

The WRN protein mutated in the hereditary premature aging disorder Werner syndrome plays a vital role in handling, processing, and restoring perturbed replication forks. One of its most abundant partners, Replication Protein A (RPA), has been shown to robustly enhance WRN helicase activity in specific cases when tested in vitro. However, the significance of RPA-binding to WRN at replication forks in vivo has remained largely unexplored. In this study, we have identified several conserved phosphorylation sites in the acidic domain of WRN that are targeted by Casein Kinase 2 (CK2). Surprisingly, these phosphorylation sites are essential for the interaction between WRN and RPA, both in vitro and in human cells. By characterizing a CK2-unphosphorylatable WRN mutant that lacks the ability to bind RPA, we have determined that the WRN-RPA complex plays a critical role in fork recovery after replication stress whereas the WRN-RPA interaction is not necessary for the processing of replication forks or preventing DNA damage when forks stall or collapse. When WRN fails to bind RPA, fork recovery is impaired, leading to the accumulation of single-stranded DNA gaps in the parental strands, which are further enlarged by the structure-specific nuclease MRE11. Notably, RPA-binding by WRN and its helicase activity are crucial for countering the persistence of G4 structures after fork stalling. Therefore, our findings reveal for the first time a novel role for the WRN-RPA interaction to facilitate fork restart, thereby minimizing G4 accumulation at single-stranded DNA gaps and suppressing accumulation of unreplicated regions that may lead to MUS81-dependent double-strand breaks requiring efficient repair by RAD51 to prevent excessive DNA damage.

cell biology↗

bioRxiv2023

WRNIP1 prevents transcription-associated genomic instability

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.

cell biology↗

bioRxiv2023

RAD52 prevents accumulation of Polalpha-dependent replication gaps at perturbed replication forks in human cells

Replication gaps can arise as a consequence of perturbed DNA replication, and their accumulation might undermine the stability of the genome. Loss of RAD52, a protein involved in the regulation of fork reversal, promotes accumulation of parental ssDNA gaps during replication perturbation. Here, we demonstrate that this is due to the engagement of Pol downstream of the extensive degradation of perturbed replication forks after their reversal and is not dependent on PrimPol. Pol is hyper-recruited at parental ssDNA in the absence of RAD52, and this recruitment is dependent on fork reversal enzymes and RAD51. Of note, we report that the interaction between Pol and RAD51 is stimulated by RAD52 inhibition, and Pol-dependent gap accumulation requires formation of the RAD51 nucleoprotein filaments. Our data indicate that the RAD51/Pol-dependent repriming is essential to support fork progression, limit DNA damage and improve viability of RAD52-deficient cells when replication is perturbed. Altogether, this study shows that RAD51/Pol-dependent repriming is a genuine fork recovery mechanism activated to overcome loss of RAD52 function at replication forks.

cell biology↗

bioRxiv2022

WRNIP1 PREVENTS G4/R-LOOP-ASSOCIATED GENOMIC INSTABILITY

Maintenance of genome integrity is essential for cell viability and depends on complete and accurate DNA replication. However, G4/R-loops may provide significant obstacles for DNA replication, as they can cause collisions between replication fork and the transcription machinery. Hence, cells require mechanisms to counteract the presence of persistent G4/R-loops, most of which remain poorly understood. Here, we demonstrate an involvement of the Werner helicase-interacting protein 1 (WRNIP1) in preventing DNA damage induced by G4/R-loop-associated transcription-replication conflicts. We discovered that the ubiquitin-binding domain of WRNIP1 is required to efficiently avoid pathological persistence of G4/R-loops upon replication stress. Also, we observed that G4s reside within R-loops and that WRNIP1 colocalises with these structures. Furthermore, WRNIP1 plays a role in restarting replication from transcription-induced fork stalling. More importantly, we characterized the interplay between WRNIP1 and the DNA helicase FANCJ in counteracting R-loop-dependent G4 formation in response to replication stress. Collectively, our findings propose a mechanisms whereby WRNIP1, contributing to stabilise FANCJ to G4 sites, mitigates the G4/R-loop-mediated transcription-replication conflicts and protects against DNA damage accumulation.

cell biology↗