Biology subjects

Palermo, V.

Publications and source records attributed to Palermo, V..

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↗

bioRxiv2019

Checkpoint defects require WRNIP1 to counteract R-loop-associated genomic instability

Conflicts between replication and transcription are common source of genome instability and many factors participate in prevention or removal of harmful R-loops. Here, we demonstrate that a WRNIP1-mediated response plays an important role in counteracting accumulation of aberrant R-loops. Using human cellular models with compromised ATR-dependent checkpoint activation, we show that WRNIP1 is stabilised in chromatin and is needed for maintaining genome integrity by mediating the ATM-dependent phosphorylation of CHK1. Furthermore, we show that loss of WRN or ATR signalling leads to accumulation of R-loop-dependent parental ssDNA, which is covered by RAD51. We demonstrate that 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 inhibition or WRNIP1 abrogation is accompanied by increased levels of genomic instability. Overall our findings reveal a novel function of WRNIP1 in preventing R-loop-driven genome instability, providing new clues to understand the way replication-transcription conflicts are resolved.

cancer biology↗

bioRxiv2019

Generation of inducible SMARCAL1 knock-down iPSC to model severe Schimke immune-osseous dysplasia reveals a link between replication stress and altered expression of master differentiation genes

The Schimke immuno-osseous dysplasia is an autosomal recessive genetic osteochondrodysplasia characterized by dysmorphism, spondyloepiphyseal dysplasia, nephrotic syndrome and frequently T cell immunodeficiency. Several hypotheses have been proposed to explain pathophysiology of the disease, however, the mechanism by which SMARCAL1 mutations cause the syndrome is elusive. Indeed, animal models of the disease are absent or useless to provide insight into the disease mechanism, since they do not recapitulate the phenotype. We generated a conditional knockdown model of SMARCAL1 in iPSCs to mimic conditions of cells with severe form the disease. Here, we characterize this model for the presence of phenotype linked to the replication caretaker role of SMARCAL1 using multiple cellular endpoints. Our data show that conditional knockdown of SMARCAL1 in human iPSCs induces replication-dependent and chronic accumulation of DNA damage triggering the DNA damage response. Furthermore, they indicate that accumulation of DNA damage and activation of the DNA damage response correlates with increased levels of R-loops and replication-transcription interference. Finally, we provide data showing that, in SMARCAL1-deficient iPSCs, DNA damage response can be maintained active also after differentiation, possibly contributing to the observed altered expression of a subset of germ layer-specific master genes. In conclusion, our conditional SMARCAL1 iPSCs may represent a powerful model where studying pathogenetic mechanisms of severe Schimke immuno-osseous dysplasia, thus overcoming the reported inability of different model systems to recapitulate the disease.

cell biology↗

bioRxiv2018

SWITCH-LIKE PHOSPHORYLATION OF WRN INTEGRATES END-RESECTION WITH REPAIR OF DSBs AT REPLICATION FORKS

Replication-dependent DNA double-strand breaks are harmful lesions preferentially repaired by homologous recombination; a process that requires processing of DNA ends to allow RAD51-mediated strand invasion. End-resection and subsequent repair are two intertwined processes, but the mechanism underlying their execution is still poorly appreciated. The WRN helicase is one of the crucial factors for the end-resection and is instrumental to select the proper repair pathway. Here, we reveal that ordered phosphorylation of WRN by the CDK1, ATM and ATR kinases define a complex regulatory layer that is essential for correct long-range end-resection connecting it to repair by homologous recombination. We establish that long-range end-resection requires an ATM-dependent phosphorylation of WRN at Ser1058 and that phosphorylation at Ser1141, together with dephosphorylation at the CDK1 site Ser1133, is needed for the correct metabolism of RAD51 foci and RAD51-dependent repair. Collectively, our findings suggest that regulation of WRN by multiple kinases functions as molecular switch to allow a timely execution of end-resection and repair at replication-dependent DNA double-strand breaks.

cell biology↗