DNA replication and replication stress response in the context of nuclear architecture

The DNA replication process needs to be coordinated with other DNA metabolism transactions and must eventually extend to the full genome, regardless of chromatin status, gene expression, secondary structures and DNA lesions. Completeness and accuracy of DNA replication are crucial to maintain genome integrity, limiting transformation in normal cells and offering targeting opportunities for proliferating cancer cells. DNA replication is thus tightly coordinated with chromatin dynamics and 3D genome architecture, and we are only beginning to understand the underlying molecular mechanisms. While much has recently been discovered on how DNA replication initiation is organised and modulated in different genomic regions and nuclear territories-the so-called "DNA replication program"-we know much less on how the elongation of ongoing replication forks and particularly the response to replication obstacles is affected by the local nuclear organisation. Also, it is still elusive how specific components of nuclear architecture participate in the replication stress response. Here, we review known mechanisms and factors orchestrating replication initiation, and replication fork progression upon stress, focusing on recent evidence linking genome organisation and nuclear architecture with the cellular responses to replication interference, and highlighting open questions and future challenges to explore this exciting new avenue of research

Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis

The role of Rad51 in an unperturbed cell cycle has been difficult to distinguish from its DNA repair function. Here, using EM to visualize replication intermediates assembled in Xenopus laevis egg extract, we show that Rad51 is required to prevent the accumulation of single-stranded DNA (ssDNA) gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading- and lagging-strand DNA synthesis. In contrast, ssDNA gaps behind forks, which are prevalent on damaged templates, result from Mre11-dependent degradation of newly synthesized DNA strands and are suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct roles for Rad51 at replication forks, demonstrating that Rad51 protects newly synthesized DNA from Mre11-dependent degradation and promotes continuous DNA synthesis

14-3-3 Proteins Regulate Exonuclease 1–Dependent Processing of Stalled Replication Forks

Replication fork integrity, which is essential for the maintenance of genome stability, is monitored by checkpoint-mediated phosphorylation events. 14-3-3 proteins are able to bind phosphorylated proteins and were shown to play an undefined role under DNA replication stress. Exonuclease 1 (Exo1) processes stalled replication forks in checkpoint-defective yeast cells. We now identify 14-3-3 proteins as in vivo interaction partners of Exo1, both in yeast and mammalian cells. Yeast 14-3-3–deficient cells fail to induce Mec1–dependent Exo1 hyperphosphorylation and accumulate Exo1–dependent ssDNA gaps at stalled forks, as revealed by electron microscopy. This leads to persistent checkpoint activation and exacerbated recovery defects. Moreover, using DNA bi-dimensional electrophoresis, we show that 14-3-3 proteins promote fork progression under limiting nucleotide concentrations. We propose that 14-3-3 proteins assist in controlling the phosphorylation status of Exo1 and additional unknown targets, promoting fork progression, stability, and restart in response to DNA replication stress

Modeling and forecasting the oil volatility index

This paper models and forecasts the crude oil ETF volatility index (OVX). Themotivation lies on the evidence that the OVX has been used in the last years as an important alternative measure to track and analyze the volatility of future oil prices. The analysis of the OVX suggests that it presents similar features to those of the daily market volatility index. The main characteristic is the long range dependence that is modeled either by autoregressive fractional integrated moving averaging (ARFIMA) models or by heterogeneous autoregressive (HAR) specifications. Regarding the latter family of models, we first propose extensions of the HAR model that are based on the net and scale measures of oil prices changes. The aim is to improve the HAR model by including predictors that better capture the impact of oil price changes on the economy. Second, we test the forecasting performance of the new proposals and benchmarks with the model confidence set (MCS) and the Generalized-AutoContouR (G-ACR) tests interms of point forecasts and density forecasting, respectively. Our main findings are as follows: the new asymmetric proposals have superior predictive ability than the heterogeneous autoregressive leverage (HARL) model under two known loss functions. Regarding density forecasting, the best model is the one that includes the scale measureas a proxy of oil price changes and considers a flexible distribution for the errors.Acknowledgements: The third author acknowledges financial support from Spanish Ministry of Economy and Competitiveness, research projects ECO2015-70331-C2-2-R and ECO2015-65701-P and from Fundação para a Ciência e a Tecnologia, grant UID/GES/00315/2013

Mineral Carbonation of CO2 in Mafic Plutonic Rocks, I—Screening Criteria and Application to a Case Study in Southwest Portugal

This article describes the screening, ranking and characterization of ultramafic and mafic rocks in southern Portugal for mineral carbonation as an alternative to conventional CO2 storage in sedimentary rocks. A set of criteria including mineralogy, structure, surface area, distance to CO2 sources, expected volume, and socioeconomic conditions was applied to screen ultramafic and mafic rock massifs in the Alentejo region, southern Portugal. Ranking of the massifs indicated that the plutonic massifs of Sines and of Torrão-Odivelas were the most promising. A characterization was made of the Sines massif, a subvolcanic massif composed mostly of gabbros and diorites, located immediately adjacent to the CO2 sources and outcropping along 300 km2 onshore and offshore. These studies confirmed that these rock samples exhibited the appropriate mineralogical and geochemical features, but also indicated that the secondary porosity provided by the fracture patterns was very small

RAD51 restricts DNA over-replication from re-activated origins

Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and genomic instability. Most of these mechanisms limit the activity of origin licensing proteins to prevent the reactivation of origins that have already been used. Here, we have investigated whether additional controls restrict the extension of re-replicated DNA in the event of origin re-activation. In a genetic screening in cells forced to re-activate origins, we found that re-replication is limited by RAD51 and enhanced by FBH1, a RAD51 antagonist. In the presence of chromatin-bound RAD51, forks stemming from re-fired origins are slowed down, leading to frequent events of fork reversal. Eventual re-initiation of DNA synthesis mediated by PRIMPOL creates ssDNA gaps that facilitate the partial elimination of re-duplicated DNA by MRE11 exonuclease. In the absence of RAD51, these controls are abrogated and re-replication forks progress much longer than in normal conditions. Our study uncovers a safeguard mechanism to protect genome stability in the event of origin reactivation

Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers

Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occur during both intergenerational transmissions and in somatic cells. Here we describe an experimental system to analyze large-scale repeat expansions in cultured human cells. It employs a shuttle plasmid that can replicate from the SV40 origin in human cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6. It also contains a selectable cassette allowing us to detect repeat expansions that accumulated in human cells upon plasmid transformation into yeast. We indeed observed massive expansions of GAA•TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAA•TTC repeats stall replication fork progression, while the frequency of repeat expansions appears to depend on proteins implicated in replication fork stalling, reversal, and restart. Locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers, which interfere with triplex formation at GAA•TTC repeats in vitro, prevented the expansion of these repeats in human cells. We hypothesize, therefore, that triplex formation by GAA•TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart

New histone supply regulates replication fork speed and PCNA unloading

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Mcl-1 deficiency in murine livers leads to nuclear polyploidisation and mitotic errors: Implications for hepatocellular carcinoma

BACKGROUND & AIMS Mcl-1, an antiapoptotic protein overexpressed in many tumours, including hepatocellular carcinoma (HCC), represents a promising target for cancer treatment. Although Mcl-1 non-apoptotic roles might critically influence the therapeutic potential of Mcl-1 inhibitors, these functions remain poorly understood. We aimed to investigate the effects of hepatic Mcl-1 deficiency (Mcl-1$^{Δhep}$) on hepatocyte ploidy and cell cycle in murine liver in vivo and the possible implications on HCC. METHODS Livers of young Mcl-1$^{Δhep}$ and wild-type (WT) mice were analysed for ploidy profile, mitotic figures, in situ chromosome segregation, gene set enrichment analysis and were subjected to two-thirds partial hepatectomy to assess Mcl-1 deficiency effect on cell cycle progression in vivo. Mcl-1$^{Δhep}$ tumours in older mice were analysed for ploidy profile, chromosomal instability, and mutational signatures via whole exome sequencing. RESULTS In young mice, Mcl-1 deficiency leads to nuclear polyploidy and to high rates of mitotic errors with abnormal spindle figures and chromosome mis-segregation along with a prolonged spindle assembly checkpoint activation signature. Chromosomal instability and altered ploidy profile are observed in Mcl-1$^{Δhep}$ tumours of old mice as well as a characteristic mutational signature of currently unknown aetiology. CONCLUSIONS Our study suggests novel non-apoptotic effects of Mcl-1 deficiency on nuclear ploidy, mitotic regulation, and chromosomal segregation in hepatocytes in vivo. In addition, the Mcl-1 deficiency characteristic mutational signature might reflect mitotic issues. These results are of importance to consider when developing anti-Mcl-1 therapies to treat cancer. IMPACT AND IMPLICATIONS Although Mcl-1 inhibitors represent promising hepatocellular carcinoma treatment, the still poorly understood non-apoptotic roles of Mcl-1 might compromise their successful clinical application. Our study shows that Mcl-1 deficiency leads to nuclear polyploidy, mitotic errors, and aberrant chromosomal segregation in hepatocytes in vivo, whereas hepatocellular tumours spontaneously induced by Mcl-1 deficiency exhibit chromosomal instability and a mutational signature potentially reflecting mitotic issues. These results have potential implications for the development of anti-Mcl-1 therapies to treat hepatocellular carcinoma, especially as hyperproliferative liver is a clinically relevant situation

PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.Spanish Ministry of Science, Innovation and Universities (grants BFU2016-80402R and BFU2019-106707-RB to JM and PGC2018.093576-B-C21 to LB, co-sponsored by EU ERDF funds), the Swiss National Science Foundation (grant 31003A_169959 to ML), and an ERC Consolidator Grant (617102 to ML