Matteo Ferrari

Functional interplay between the 53BP1-ortholog Rad9 and the Mre11 complex regulates resection, end-tethering and repair of a double-strand break.

The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.

Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

The RSC chromatin-remodeling complex influences mitotic exit and adaptation to the spindle assembly checkpoint by controlling the Cdc14 phosphatase

Rsc2 promotes Cdc14 release from the nucleolus to free cells from mitotic arrest.

Slx4 and Rtt107 control checkpoint signalling and DNA resection at double-strand breaks

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5′ strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.

Alpha-Lipoic Acid and Antioxidant Diet Help to Improve Endothelial Dysfunction in Adolescents with Type 1 Diabetes: A Pilot Trial

After evaluating the prevalence of early endothelial dysfunction, as measured by means of reactive hyperemia in adolescents with type 1 diabetes, we started a 6-month, double-blind, randomized trial to test the efficacy of an antioxidant diet (± alpha-lipoic acid supplementation) to improve endothelial dysfunction. Seventy-one children and adolescents, ages 17 ± 3.9 yrs, with type 1 diabetes since 9.5 ± 5.3 yrs, using intensified insulin therapy, were randomized into 3 arms: (a) antioxidant diet 10.000 ORAC + alpha-lipoic acid; (b) antioxidant diet 10.000 ORAC + placebo; (c) controls. BMI, blood pressure, fasting lipid profile, HbA1c, insulin requirement, dietary habits, and body composition were determined in each patient. An antioxidant diet significantly improved endothelial dysfunction when supplemented with alpha-lipoic acid, unlike diet with placebo or controls. A significant reduction in bolus insulin was also observed. We speculate that alpha-lipoic acid might have an antioxidant effect in pediatric diabetes patients by reducing insulin.

Reduced kinase activity of polo kinase Cdc5 affects chromosome stability and DNA damage response in S. cerevisiae.

ABSTRACT Polo-like kinases (PLKs) control several aspects of eukaryotic cell division and DNA damage response. Remarkably, PLKs are overexpressed in several types of cancer, being therefore a marker of bad prognosis. As such, specific PLK kinase activity inhibitors are already used in clinical trials and the regulation of PLK activation is a relevant topic of cancer research. Phosphorylation of threonine residues in the T-loop of the kinase domain is pivotal for PLKs activation. Here, we show that T238A substitution in the T-loop reduces the kinase activity of Cdc5, the only PLK in Saccharomyces cerevisiae, with minor effect on cell growth in unperturbed conditions. However, the cdc5-T238A cells have increased rate of chromosome loss and gross chromosomal rearrangements, indicating altered genome stability. Moreover, the T238A mutation affects timely localization of Cdc5 to the spindle pole bodies and blocks cell cycle restart after one irreparable double-strand break. In cells responding to alkylating agent metylmethane sulfonate (MMS), the cdc5-T238A mutation reduces the phosphorylation of Mus81-Mms4 resolvase and exacerbates the MMS sensitivity of sgs1Δ cells that accumulate Holliday junctions. Of importance, the previously described checkpoint adaptation defective allele, cdc5-ad does not show reduced kinase activity, defective Mms4 phosphorylation and genetic interaction with sgs1Δ. Our data define the importance of regulating Cdc5 activity through T-loop phosphorylation to preserve genome integrity and respond to DNA damage.

Tid1/Rdh54 translocase is phosphorylated through a Mec1- and Rad53-dependent manner in the presence of DSB lesions in budding yeast.

Saccharomyces cerevisiae cells with a single double-strand break (DSB) activate the ATR/Mec1-dependent checkpoint response as a consequence of extensive ssDNA accumulation. The recombination factor Tid1/Rdh54, a member of the Swi2-like family proteins, has an ATPase activity and may contribute to the remodelling of nucleosomes on DNA. Tid1 dislocates Rad51 recombinase from dsDNA, can unwind and supercoil DNA filaments, and has been implicated in checkpoint adaptation from a G2/M arrest induced by an unrepaired DSB. Here we show that both ATR/Mec1 and Chk2/Rad53 kinases are implicated in the phosphorylation of Tid1 in the presence of DNA damage, indicating that the protein is regulated during the DNA damage response. We show that Tid1 ATPase activity is dispensable for its phosphorylation and for its recruitment near a DSB, but it is required to switch off Rad53 activation and for checkpoint adaptation. Mec1 and Rad53 kinases, together with Rad51 recombinase, are also implicated in the hyper-phosphorylation of the ATPase defective Tid1-K318R variant and in the efficient binding of the protein to the DSB site. In summary, Tid1 is a novel target of the DNA damage checkpoint pathway that is also involved in checkpoint adaptation.

Risky Behaviours in Teens with Type 1 Diabetes: New Ideas for Research Regarding a Not Too Studied Topic

Risk is the potential of gaining or losing something of value. Values (such as physical health, social status, emotional well-being or financial wealth) can be gained or lost when taking risk resulting from a given action or inaction. Risk can also be defined as the intentional interaction with uncertainty. When talking about health, especially during adolescence, the expression risky behaviour can be defined as the participation in activities that may compromise the physical and mental health of adolescents. Many of these behaviours can be started simply by the explorative character of youths or by the influence of the youths’ environment (peers, family); moreover, if risky behaviour is not identified early, it can lead to the consolidation of these attitudes with significant individual, family and social consequences.

Nutritional Aspects of Type 1 Diabetes: We Need to Keep Struggling Against Palaeolithic Diet (How Research Helps Us to Do the Right Thing)

For both type 1 and type 2 diabetes, eating healthy is key to blood glucose management [1]. Nowadays it is well recognised that, for each child and adolescent with type 1 diabetes, nutrition therapy is recommended. Implementation of an individualised meal plan with appropriate insulin adjustments can help to improve glycaemic control. The optimal macronutrient distribution varies depending on an individualised assessment [2].

Sensor-augmented pump and Down syndrome: a new tool in tricky patients

W e read with great interest the paper by Piccini and cols. (1) published in the July issue of this Journal. Some years ago, we published the first report ever (to the best of our knowledge) of successful treatment of a girl with Down syndrome, Hashimoto’s thyroiditis and celiac disease with continuous subcutaneous insulin infusion (2). Since then, her glycemic control was kept constant and, most of the time, in the target range (HbA1c in 2009: 7.75 ± 0.21%; HbA1c in 2010: 7.35 ± 0.19%; HbA1c in 2011: 7.42 ± 0.30%). At the end of 2011, sensor-augmented pump was initiated (Animas® VibeTM, West Chester, PA, USA) because of both a quite high glycemic variability and the parents’ request, and her HbA1c kept improving (HbA1c in 2012: 7.30 ± 0.20%; HbA1c in 2013: 7.10 ± 0.28%). CSII has been recognized as effective and safe in pediatric (3) and in adult patients (4), not only in the short run, but even after many years (5). In patients with Down syndrome and type 1 diabetes, glycemic control may sometimes be particularly tricky (6,7). In our patient, as well as in the one of Piccini and cols. (1), CSII was a safe and effective way to manage diabetes. For a successful CSII therapy in a patient with Down syndrome, whose mental function may be impaired, the collaboration of a highly motivated and compliant family is essential, as well as a skilled multidisciplinary diabetes team (8). Given all of this, pump increased the patient’s and family’s flexibility, as we had previously reported (2). The significant improvement in the glycemic control observed, and the high level of acceptance of CSII therapy observed in both our case and in that of Piccini and cols. is worth the effort of the patient’s family and of the diabetes team in ensuring that the patient has a flexible life. Perhaps CSII therapy might be taken into account when considering insulin therapy in patients with Down’s syndrome.