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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.

Franchitto A.,Section of Molecular Epidemiology
BioMed Research International | Year: 2013

Common fragile sites (CFS) are heritable nonrandomly distributed loci on human chromosomes that exhibit an increased frequency of chromosomal breakage under conditions of replication stress. They are considered the preferential targets for high genomic instability from the earliest stages of human cancer development, and increased chromosome instability at these loci has been observed following replication stress in a subset of human genetic diseases. Despite their biological and medical relevance, the molecular basis of CFS fragility in vivo has not been fully elucidated. At present, different models have been proposed to explain how instability at CFS arises and multiple factors seem to contribute to their instability. However, all these models involve DNA replication and suggest that replication fork stalling along CFS during DNA synthesis is a very frequent event. Consistent with this, the maintenance of CFS stability relies on the ATR-dependent checkpoint, together with a number of proteins promoting the recovery of stalled replication forks. In this review, we discuss mainly the possible causes that threaten the integrity of CFS in the light of new findings, paying particular attention to the role of the S-phase checkpoint. © 2013 Annapaola Franchitto.

Gunn D.A.,Unilever | De Craen A.J.M.,Leiden University | Dick J.L.,Unilever | Tomlin C.C.,Unilever | And 9 more authors.
Journals of Gerontology - Series A Biological Sciences and Medical Sciences | Year: 2013

Background.As facial appearance can be readily quantified and skin tissue easily accessed, they could be valuable tools for determining how biological mechanisms influence tissue degeneration with age and, consequently, human health and lifespan. It is unknown, however, whether appearance reflects disease risk or lifespan independently of factors already known to associate with both health and appearance.Methods.In a cross-sectional study, we compared the amount of skin wrinkling on a sun-protected site (upper inner arm) and the facial appearance of 261 offspring (mean age 63.2 y) of nonagenarian siblings with 253 age-matched controls (mean age 62.7 y), all with no reported disease history. We next examined whether any appearance features that significantly associated with familial longevity also associated with the Framingham cardiovascular disease (CVD) risk score. All analyses were adjusted for chronological age, smoking, photodamage, and body mass index.Results.Female and male offspring had reduced upper inner arm skin wrinkling (p =. 03 and p <. 001, respectively), and the male offspring looked 1.4 y younger than the controls (p =. 002). There were no significant associations between CVD risk and upper inner arm skin wrinkling. Women in the lowest quartile of CVD risk looked more than 2 y younger for their age than those in higher risk quartiles (p =. 002). Systolic blood pressure was the most significant (p =. 004) CVD risk factor that was associated with perceived age in women.Conclusions.Facial appearance and skin wrinkling at a sun-protected site reflect the propensity to reach an extreme old age, and facial appearance reflects the risk of succumbing to CVD independently of chronological age, smoking, photodamage, and BMI. © 2012 The Author. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved.

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.

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

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