A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells.

Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells.

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end joining.

The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ.

Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases.

Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5' of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols ß and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.

From R-Loops to G-Quadruplexes: Emerging New Threats for the Replication Fork.

Replicating the entire genome is one of the most complex tasks for all organisms. Research carried out in the last few years has provided us with a clearer picture on how cells preserve genomic information from the numerous insults that may endanger its stability. Different DNA repair pathways, coping with exogenous or endogenous threat, have been dissected at the molecular level. More recently, there has been an increasing interest towards intrinsic obstacles to genome replication, paving the way to a novel view on genomic stability. Indeed, in some cases, the movement of the replication fork can be hindered by the presence of stable DNA: RNA hybrids (R-loops), the folding of G-rich sequences into G-quadruplex structures (G4s) or repetitive elements present at Common Fragile Sites (CFS). Although differing in their nature and in the way they affect the replication fork, all of these obstacles are a source of replication stress. Replication stress is one of the main hallmarks of cancer and its prevention is becoming increasingly important as a target for future chemotherapeutics. Here we will try to summarize how these three obstacles are generated and how the cells handle replication stress upon their encounter. Finally, we will consider their role in cancer and their exploitation in current chemotherapeutic approaches.

Gene Expression Profiles Controlled by the Alternative Splicing Factor Nova2 in Endothelial Cells.

Alternative splicing (AS) plays an important role in expanding the complexity of the human genome through the production of specialized proteins regulating organ development and physiological functions, as well as contributing to several pathological conditions. How AS programs impact on the signaling pathways controlling endothelial cell (EC) functions and vascular development is largely unknown. Here we identified, through RNA-seq, changes in mRNA steady-state levels in ECs caused by the neuro-oncological ventral antigen 2 (Nova2), a key AS regulator of the vascular morphogenesis. Bioinformatics analyses identified significant enrichment for genes regulated by peroxisome proliferator-activated receptor-gamma (Ppar-γ) and E2F1 transcription factors. We also showed that Nova2 in ECs controlled the AS profiles of Ppar-γ and E2F dimerization partner 2 (Tfdp2), thus generating different protein isoforms with distinct function (Ppar-γ) or subcellular localization (Tfdp2). Collectively, our results supported a mechanism whereby Nova2 integrated splicing decisions in order to regulate Ppar-γ and E2F1 activities. Our data added a layer to the sequential series of events controlled by Nova2 in ECs to orchestrate vascular biology.

UBR5 interacts with the replication fork and protects DNA replication from DNA polymerase η toxicity.

Accurate DNA replication is critical for the maintenance of genome integrity and cellular survival. Cancer-associated alterations often involve key players of DNA replication and of the DNA damage-signalling cascade. Post-translational modifications play a fundamental role in coordinating replication and repair and central among them is ubiquitylation. We show that the E3 ligase UBR5 interacts with components of the replication fork, including the translesion synthesis (TLS) polymerase polη. Depletion of UBR5 leads to replication problems, such as slower S-phase progression, resulting in the accumulation of single stranded DNA. The effect of UBR5 knockdown is related to a mis-regulation in the pathway that controls the ubiquitylation of histone H2A (UbiH2A) and blocking this modification is sufficient to rescue the cells from replication problems. We show that the presence of polη is the main cause of replication defects and cell death when UBR5 is silenced. Finally, we unveil a novel interaction between polη and H2A suggesting that UbiH2A could be involved in polη recruitment to the chromatin and the regulation of TLS.

Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing.

The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1-inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK-dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1-deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1-depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition.

Performance output data and configurations of stencil compilers experiments run through PROVA!

The data in this article are related to the research article titled "Reproducible Stencil Compiler Benchmarks Using PROVA!". Stencil kernels have been implemented using a naïve OpenMP (OpenMP Architecture Review Board, 2016) [1] parallelization and then using the stencil compilers PATUS (Christen et al., 2011) [2] and (Bondhugula et al., 2008) PLUTO [3]. Performance experiments have been run on different architectures, by using PROVA! (Guerrera et al., 2017) [4], a distributed workflow and system management tool to conduct reproducible research in computational sciences. Information like version of the compiler, compilation flags, configurations, experiment parameters and raw results are fundamental contextual information for the reproducibility of an experiment. All this information is automatically stored by PROVA! and, for the experiments presented in this paper, are available at https://github.com/sguera/FGCS17.

Phosphorylation regulates human polη stability and damage bypass throughout the cell cycle.

DNA translesion synthesis (TLS) is a crucial damage tolerance pathway that oversees the completion of DNA replication in the presence of DNA damage. TLS polymerases are capable of bypassing a distorted template but they are generally considered inaccurate and they need to be tightly regulated. We have previously shown that polη is phosphorylated on Serine 601 after DNA damage and we have demonstrated that this modification is important for efficient damage bypass. Here we report that polη is also phosphorylated by CDK2, in the absence of damage, in a cell cycle-dependent manner and we identify serine 687 as an important residue targeted by the kinase. We discover that phosphorylation on serine 687 regulates the stability of the polymerase during the cell cycle, allowing it to accumulate in late S and G2 when productive TLS is critical for cell survival. Furthermore, we show that alongside the phosphorylation of S601, the phosphorylation of S687 and S510, S512 and/or S514 are important for damage bypass and cell survival after UV irradiation. Taken together our results provide new insights into how cells can, at different times, modulate DNA TLS for improved cell survival.

The Regulation of DNA Damage Tolerance by Ubiquitin and Ubiquitin-Like Modifiers.

DNA replication is an extremely complex process that needs to be executed in a highly accurate manner in order to propagate the genome. This task requires the coordination of a number of enzymatic activities and it is fragile and prone to arrest after DNA damage. DNA damage tolerance provides a last line of defense that allows completion of DNA replication in the presence of an unrepaired template. One of such mechanisms is called post-replication repair (PRR) and it is used by the cells to bypass highly distorted templates caused by damaged bases. PRR is extremely important for the cellular life and performs the bypass of the damage both in an error-free and in an error-prone manner. In light of these two possible outcomes, PRR needs to be tightly controlled in order to prevent the accumulation of mutations leading ultimately to genome instability. Post-translational modifications of PRR proteins provide the framework for this regulation with ubiquitylation and SUMOylation playing a pivotal role in choosing which pathway to activate, thus controlling the different outcomes of damage bypass. The proliferating cell nuclear antigen (PCNA), the DNA clamp for replicative polymerases, plays a central role in the regulation of damage tolerance and its modification by ubiquitin, and SUMO controls both the error-free and error-prone branches of PRR. Furthermore, a significant number of polymerases are involved in the bypass of DNA damage possess domains that can bind post-translational modifications and they are themselves target for ubiquitylation. In this review, we will focus on how ubiquitin and ubiquitin-like modifications can regulate the DNA damage tolerance systems and how they control the recruitment of different proteins to the replication fork.