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Leuzzi G.,Section of Molecular Epidemiology | Marabitti V.,Section of Molecular Epidemiology | Pichierri P.,Section of Experimental and Computational Carcinogenesis | Franchitto A.,Section of Molecular Epidemiology
EMBO Journal | Year: 2016

Accurate handling of stalled replication forks is crucial for the maintenance of genome stability. RAD51 defends stalled replication forks from nucleolytic attack, which otherwise can threaten genome stability. However, the identity of other factors that can collaborate with RAD51 in this task is poorly elucidated. Here, we establish that human Werner helicase interacting protein 1 (WRNIP1) is localized to stalled replication forks and cooperates with RAD51 to safeguard fork integrity. We show that WRNIP1 is directly involved in preventing uncontrolled MRE11-mediated degradation of stalled replication forks by promoting RAD51 stabilization on ssDNA. We further demonstrate that replication fork protection does not require the ATPase activity of WRNIP1 that is however essential to achieve the recovery of perturbed replication forks. Loss of WRNIP1 or its catalytic activity causes extensive DNA damage and chromosomal aberrations. Intriguingly, downregulation of the anti-recombinase FBH1 can compensate for loss of WRNIP1 activity, since it attenuates replication fork degradation and chromosomal aberrations in WRNIP1-deficient cells. Therefore, these findings unveil a unique role for WRNIP1 as a replication fork-protective factor in maintaining genome stability. © 2016 The Authors

Franchitto A.,Section of Molecular Epidemiology | Franchitto A.,Genome Stability Group | Pichierri P.,Section of Experimental and Computational Carcinogenesis | Pichierri P.,Genome Stability Group
Cellular and Molecular Life Sciences | Year: 2014

The acquisition of genomic instability is a triggering factor in cancer development, and common fragile sites (CFS) are the preferential target of chromosomal instability under conditions of replicative stress in the human genome. Although the mechanisms leading to CFS expression and the cellular factors required to suppress CFS instability remain largely undefined, it is clear that DNA becomes more susceptible to breakage when replication is impaired. The models proposed so far to explain how CFS instability arises imply that replication fork progression along these regions is perturbed due to intrinsic features of fragile sites and events that directly affect DNA replication. The observation that proteins implicated in the safe recovery of stalled forks or in engaging recombination at collapsed forks increase CFS expression when downregulated or mutated suggests that the stabilization and recovery of perturbed replication forks are crucial to guarantee CFS integrity. © 2014 Springer Basel.

Pichierri P.,Genome Stability Group | Ammazzalorso F.,Genome Stability Group | Bignami M.,Section of Experimental and Computational Carcinogenesis | Franchitto A.,Genome Stability Group
Aging | Year: 2011

The Werner syndrome protein (WRN) is a member of the human RecQ family DNA helicases implicated in the maintenance of genome stability. Loss of WRN gives rise to the Werner syndrome, a genetic disease characterised by premature aging and cancer predisposition. WRN plays a crucial role in the response to replication stress and significantly contributes to the recovery of stalled replication forks, although how this function is regulated is not fully appreciated. There is a growing body of evidence that WRN accomplishes its task in close connection with the replication checkpoint. In eukaryotic cells, the replication checkpoint response, which involves both the ATR and ATM kinase activities, is deputed to the maintenance of fork integrity and re-establishment of fork progression. Our recent findings indicate that ATR and ATM modulate WRN function at defined steps of the response to replication fork arrest. This review focuses on the novel evidence of a functional relationship between WRN and the replication checkpoint and how this cross-talk might contribute to prevent genome instability, a common feature of senescent and cancer cells. © Pichierri.

Iannascoli C.,Section of Experimental and Computational Carcinogenesis | Palermo V.,Section of Experimental and Computational Carcinogenesis | Murfuni I.,Section of Experimental and Computational Carcinogenesis | Franchitto A.,Section of Molecular Epidemiology | Pichierri P.,Section of Experimental and Computational Carcinogenesis
Nucleic Acids Research | Year: 2015

The WRN helicase/exonuclease protein is required for proper replication fork recovery and maintenance of genome stability. However, whether the different catalytic activities of WRN cooperate to recover replication forks in vivo is unknown. Here, we show that, in response to replication perturbation induced by low doses of the TOP1 inhibitor camptothecin, loss of the WRN exonuclease resulted in enhanced degradation and ssDNA formation at nascent strands by the combined action of MRE11 and EXO1, as opposed to the limited processing of nascent strands performed by DNA2 in wild-type cells. Nascent strand degradation by MRE11/EXO1 took place downstream of RAD51 and affected the ability to resume replication, which correlated with slow replication rates in WRN exonuclease-deficient cells. In contrast, loss of the WRN helicase reduced exonucleolytic processing at nascent strands and led to severe genome instability. Our findings identify a novel role of the WRN exonuclease at perturbed forks, thus providing the first in vivo evidence for a distinct action of the two WRN enzymatic activities upon fork stalling and providing insights into the pathological mechanisms underlying the processing of perturbed forks. © 2015 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research.

Lupari E.,Section of Molecular Epidemiology | Ventura I.,Section of Experimental and Computational Carcinogenesis | Marcon F.,Section of Genetic Toxicology | Aquilina G.,Section of Experimental and Computational Carcinogenesis | And 2 more authors.
DNA Repair | Year: 2012

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O 6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O 6-methylguanine (O 6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O 6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O 6-meGua facilitated by RAD 51 binding. © 2012 Elsevier B.V.

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