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Abstract The ROBoCoP project is launched within the EU COST Action CA16113 “CliniMARK” aiming to increase the number of clinically validated biomarkers and focused on chronic obstructive pulmonary disease (COPD) biomarker development and validation. ROBoCoP encompasses two consecutive studies consisting of a pilot study followed by a field study. The pilot study is a longitudinal exposure assessment and biomarker study aiming at: 1-understanding the suitability of the candidate biomarkers in surveying populations at risk such as workers exposed to COPD causing agents; 2-determining the best sampling plan with respect to the half-life of the candidate biomarkers; 3-implementing and validating the sampling procedures and analytical methods; 4-selecting the best suitable biomarkers to be measured in the field. Each study participant is surveyed every day during the 6–8 h work-shifts for two consecutive weeks. The field study has an implementation research designe that enabled us to demonstrate the applicability of the standardized protocol for biomarker measurements in occupational settings while also assessing the biomarkers’ validity. ROBoCoP will focus on particulate matter (PM) exposure measurements, exposure biomarkers and a series of effect biomarkers, including markers of lipoperoxidation: 8-isoprostane, malondialdehyd in exhaled breath condensate (EBC) and urine, potential markers of nitrosative stress: NO2 −, NO3 − and formate anion in EBC; markers of DNA oxidation: 8-hydroxy-2’deoxyguanosine in EBC and urine, marker of genotoxicity: micronuclei in buccal cells, and oxidative potential in exhaled air (OPEA). OPEA appears particularly promising as a clinical biomarker for detecting COPD, and will be tested independently and as part of a biomarker panel. COPD diagnosis will be performed by an experienced occupational physician according to international diagnostic standards and confirmed by a pulmonologist. This research will include approximatively 300 underground subway workers randomly selected from the personnel registry of a large Parisian transport company. Underground subways are suggested as the most PM polluted urban transport environment. We believe this occupational exposure is relevant for biomonitoring of workers and early detection of respiratory diseases.

Abstract Background Vibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium. Results In this study, we present a genome-wide CRISPR-dCas9 screen specifically optimized for the N16961 El Tor model strain of V. cholerae. This approach is characterized by a tight control of dCas9 expression and activity, as well as a streamlined experimental setup. Our library allows the depletion of 3,674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for genes that are essential during exponential growth in rich medium and identified 369 genes for which guides were significantly depleted from the library (log2FC < -2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens. Conclusion We thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae.

Abstract Background Chronic obstructive pulmonary disease (COPD) affecting 334 million people in the world remains a major cause of morbidity and mortality. Proper diagnosis of COPD is still a challenge and largely solely based on spirometric criteria. We aimed to investigate the potential of nitrosative/oxidative stress and related metabolic biomarkers in exhaled breath condensate (EBC) to discriminate COPD patients. Methods Three hundred three participants were randomly selected from a 15,000-transit worker cohort within the Respiratory disease Occupational Biomonitoring Collaborative Project (ROBoCoP). COPD was defined using the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria as post-bronchodilator ratio of Forced Expiratory Volume in 1st second to Forced Vital Capacity

Linear unconstrained DNA cannot harbor supercoils since these supercoils can diffuse and be eliminated by free rotation of the DNA strands at the end of the molecule. Mammalian telomeres, despite constituting the ends of linear chromosomes, can hold supercoils and be subjected to topological stress. While negative supercoiling was previously observed, thus proving the existence of telomeric topological constraints, positive supercoils were never probed due to the lack of an appropriate tool. Indeed, the few tools available currently could only investigate unwound (Trioxsalen) or overwound (GapR) DNA topology (variations in twist) but not the variations in writhe (supercoils and plectonemes). To address this question, we have designed innovative tools aimed at analyzing both positive and negative DNA writhe in cells. Using them, we could observe the build-up of positive supercoils following replication stress and inhibition of Topoisomerase 2 on telomeres. TRF2 depletion caused both telomere relaxation and an increase in positive supercoils while the inhibition of Histone Deacetylase I and II by TSA only caused telomere relaxation. Moving outside telomeres, we also observed a build-up of positive supercoils on the FRA3B fragile site following replication stress, suggesting a topological model of DNA fragility for this site.

Introduction: Health effects after long-term exposure to subway particulate matter (PM) remain unknown due to the lack of individual PM exposure data. This study aimed to apply the job exposure matrix (JEM) approach to retrospectively assess occupational exposure to PM in the Parisian subway. Methods: Job, the line and sector of the transport network, as well as calendar period were four JEM dimensions. For each combination of these dimensions, we generated statistical models to estimate the annual average PM10 concentration using data from an exhaustive inventory of the PM measurement campaigns conducted between 2004 and 2020 in the Parisian subway and historical data from the Parisian air pollution monitoring network. The resulting JEM and its exposure estimates were critically examined by experts using the uncertainty analysis framework. Results: The resulting JEM allows for the assignment of the estimated annual PM10 concentration to three types of professionals working in the subway: locomotive operators, station agents, and security guards. The estimates’ precision and validity depend on the amount and quality of PM10 measurement data used in the job-, line-, and sector-specific models. Models using large amounts of personal exposure measurement data produced rather robust exposure estimates compared to models with lacunary data (i.e., in security guards). The analysis of uncertainty around the exposure estimates allows for the identification of the sources of uncertainty and parameters to be addressed in the future in order to refine and/or improve the JEM. Conclusions: The JEM approach seems relevant for the retrospective exposure assessment of subway workers. When applied to available data on PM10, it allows for the estimation of this exposure in locomotive operators and station agents with an acceptable validity. Conversely, for security guards, the current estimates have insufficient validity to recommend their use in an epidemiological study. Therefore, the current JEM should be considered as a valid prototype, which shall be further improved using more robust measurements for some jobs. This JEM can also be further refined by considering additional exposure determinants.

Abstract Background Underground transportation systems can contribute to the daily particulates and metal exposures for both commuter and subway workers. The redox and metabolic changes in workers exposed to such metal-rich particles have yet to be characterized. We hypothesize that the distribution of nitrosative/oxidative stress and related metabolic biomarkers in exhaled breath condensate (EBC) are modified depending on exposures. Results Particulate number and size as well as mass concentration and airborne metal content were measured in three groups of nine subway workers (station agents, locomotive operators and security guards). In parallel, pre- and post-shift EBC was collected daily during two consecutive working weeks. In this biological matrix, malondialdehyde, lactate, acetate, propionate, butyrate, formate, pyruvate, the sum of nitrite and nitrate (ΣNOx) and the ratio nitrite/nitrate as well as metals and nanoparticle concentrations was determined. Weekly evolution of the log-transformed selected biomarkers as well as their association with exposure variables was investigated using linear mixed effects models with the participant ID as random effect. The professional activity had a strong influence on the pattern of anions and malondialdehyde in EBC. The daily number concentration and the lung deposited surface area of ultrafine particles was consistently and mainly associated with nitrogen oxides variations during the work-shift, with an inhibitory effect on the ΣNOx. We observed that the particulate matter (PM) mass was associated with a decreasing level of acetate, lactate and ΣNOx during the work-shift, suggestive of a build-up of these anions during the previous night in response to exposures from the previous day. Lactate was moderately and positively associated with some metals and with the sub-micrometer particle concentration in EBC. Conclusions These results are exploratory but suggest that exposure to subway PM could affect concentrations of nitrogen oxides as well as acetate and lactate in EBC of subway workers. The effect is modulated by the particle size and can correspond to the body’s cellular responses under oxidative stress to maintain the redox and/or metabolic homeostasis.

Linear unconstrained DNA cannot harbor supercoils since these supercoils can diffuse and be eliminated by free rotation of the DNA strands at the end of the molecule. Mammalian telomeres, despite constituting the ends of linear chromosomes, can hold supercoils and be subjected to topological stress. While negative supercoiling was previously observed, thus proving the existence of telomeric topological constraints, positive supercoils were never probed due to the lack of an appropriate tool. Indeed, the few tools available currently could only investigate unwound (Trioxsalen) or overwound (GapR) DNA topology (variations in twist) but not the variations in writhe (supercoils and plectonemes). To address this question, we have designed innovative tools aimed at analyzing both positive and negative DNA writhe in cells. Using them, we could observe the build-up of positive supercoils following replication stress and inhibition of Topoisomerase 2 on telomeres. TRF2 depletion caused both telomere relaxation and an increase in positive supercoils while the inhibition of Histone Deacetylase I and II by TSA only caused telomere relaxation. Moving outside telomeres, we also observed a build-up of positive supercoils on the FRA3B fragile site following replication stress, suggesting a topological model of DNA fragility for this site. [ABSTRACT FROM AUTHOR]

The ischemic penumbra is defined as the severely hypoperfused, functionally impaired, at-risk but not yet infarcted tissue that will be progressively recruited into the infarct core. Early reperfusion aims to save the ischemic penumbra by preventing infarct core expansion and is the mainstay of acute ischemic stroke therapy. Intravenous thrombolysis and mechanical thrombectomy for selected patients with large vessel occlusion has been shown to improve functional outcome. Given the varying speed of infarct core progression among individuals, a therapeutic window tailored to each patient has recently been proposed. Recent studies have demonstrated that reperfusion therapies are beneficial in patients with a persistent ischemic penumbra, beyond conventional time windows. As a result, mapping the penumbra has become crucial in emergency settings for guiding personalized therapy. The penumbra was first characterized as an area with a reduced cerebral blood flow, increased oxygen extraction fraction and preserved cerebral metabolic rate of oxygen using positron emission tomography (PET) with radiolabeled O2. Because this imaging method is not feasible in an acute clinical setting, the magnetic resonance imaging (MRI) mismatch between perfusion-weighted imaging and diffusion-weighted imaging, as well as computed tomography perfusion have been proposed as surrogate markers to identify the penumbra in acute ischemic stroke patients. Transversal studies comparing PET and MRI or using longitudinal assessment of a limited sample of patients have been used to define perfusion thresholds. However, in the era of mechanical thrombectomy, these thresholds are debatable. Using various MRI methods, the original penumbra definition has recently gained a significant interest. The aim of this review is to provide an overview of the evolution of the ischemic penumbra imaging methods, including their respective strengths and limitations, as well as to map the current intellectual structure of the field using bibliometric analysis and explore future directions.

Background: Air pollution in subway environments is a growing concern as it often exceeds WHO recommendations for indoor air quality. Ultrafine particles (UFP), for which there is still no regulation nor a standardized exposure monitoring method, are the strongest contributor to this pollution when the number concentration is used as exposure metric. Objectives: We aimed to assess the real-time UFP number concentration in the personal breathing zone (PBZ) of three types of underground Parisian subway professionals and analyze it using a novel Bayesian spline approach. Consecutively, we investigated the effect of job, week day, subway station, worker location, and some further events on UFP number concentrations. Methods: The data collection procedure originated from a longitudinal study and lasted for a total duration of 6 weeks (from October 7 to November 15, 2019, i.e. two weeks per type of subway professionals). Time-series were built from the real-time particle number concentration (PNC) measured in the PBZ of professionals during their work-shifts. Complementarily, contextual information expressed as Station, Environment, and Event variables were extracted from activity logbooks completed for every work-shift. A Bayesian spline approach was applied to model the PNC within a Bayesian framework as a function of the mentioned contextual information. Results: Overall, the Bayesian spline method suited a real-time personal PNC data modeling approach. The model enabled estimating the differences in UFP exposure between subway professionals, stations, and various locations. Our results suggest a higher PNC closer to the subway tracks, with the highest PNC on subway station platforms. Studied event and week day variables had a lesser influence. Conclusion: It was shown that the Bayesian spline method is suitable to investigate individual exposure to UFP in underground subway settings. This method is informative for better documenting the magnitude and variability of UFP exposure, and for understanding the determinants in view of further regulation and control of this exposure.

Background: One of the main challenges for clinicians is to ensure that alcohol withdrawal treatment is the most effective possible after discharge. To address this issue, we designed a pilot study to investigate the efficacy of the rehabilitation treatment on the main stages of information processing, using an electroencephalographic method. This topic is of main importance as relapse rates after alcohol withdrawal treatment remain very high, indicating that established treatment methods are not fully effective in all patients in the long run.Method: We examined in alcohol-dependent patients (ADP) the effects of the benzodiazepine-based standard detoxification program on event-related potential components at incoming (D0) and completion (D15) of the treatment, using tasks of increasing difficulty (with and without workload) during an auditory oddball target paradigm. Untreated non-alcohol-dependent-volunteers were used as matching controls.Results: At D0, ADP displayed significantly lower amplitude for all ERP components in both tasks, as compared to controls. At D15, this difference disappeared for the amplitude of the N1 component during the workload-free task, as well as the amplitude of the P3b for both tasks. Meanwhile, the amplitude of the N2 remained lower in both tasks for ADP. At D0, latencies of N2 and P3b in both task conditions were longer in ADP, as compared to controls, whilst the latency of N1 was unchanged. At D15, the N2 latency remained longer for the workload condition only, whereas the P3b latency remained longer for the workload-free task only.Conclusion: The present pilot results provide evidence for a persistence of impaired parameters of ERP components, especially the N2 component. This suggests that neural networks related to attention processing remain dysfunctional. Longitudinal long-term follow-up of these patients is mandatory for further assessment of a link between ERP alterations and a later risk of relapse.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site‐specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21‐bp core‐attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7‐bp inverted regions corresponding to mv4Int core‐binding sites surrounding a 7‐bp strand‐exchange region. We also examined the different compositional constraints in the core‐binding regions, which define the sequence space of permissible recombination sites. [ABSTRACT FROM AUTHOR]

Common Fragile Sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Here the authors use genome-wide and single cell techniques to assess how replication timing and transcriptional activity correlate with genome stability.

The regularly reported associations between particulate matter (PM) exposure, and morbidity and mortality due to respiratory, cardiovascular, cancer, and metabolic diseases have led to the reduction in recommended outdoor PM10 and PM2.5 exposure limits. However, indoor PM10 and PM2.5 concentrations in subway systems in many cities are often higher than outdoor concentrations. The effects of these exposures on subway workers and passengers are not well known, mainly because of the challenges in exposure assessment and the lack of longitudinal studies combining comprehensive exposure and health surveillance. To fulfill this gap, we made an inventory of the PM measurement campaigns conducted in the Parisian subway since 2004. We identified 5856 PM2.5 and 18,148 PM10 results from both personal and stationary air sample measurements that we centralized in a database along with contextual information of each measurement. This database has extensive coverage of the subway network and will enable descriptive and analytical studies of indoor PM exposure in the Parisian subway and its potential effects on human health.

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.

A study published in the journal Clinical & Experimental Allergy investigated the connection between long-term exposure to particulate matter (PM10) in subway environments and the prevalence of asthma in subway workers. The study discovered that there was a link between the concentration of nitrite in exhaled breath condensate (EBC) and atopic sensitization, poly-atopic sensitization, and current asthma. Nitrate levels, on the other hand, were negatively associated with current eczema. The study used a retrospective exposure assessment method and had a high response rate, but it had limitations such as a small sample size and reliance on self-reported data. The study emphasizes the risk of allergic asthma in subway settings and the potential use of noninvasive biomarkers in occupational allergology research. [Extracted from the article]

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant‐negative form of RAD51 (SMRad51) that suppresses RAD51‐mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti‐tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging. Synopsis: Mutations of homologous recombination factors are linked to tumorigenesis, but whether this is also the case for RAD51 remains unclear. A new transgenic mouse model expressing an inducible dominant‐negative form, SMRAD51, reveals a novel postnatal pro‐aging but anti‐tumorigenic effect of functional inhibition of RAD51 in vivo. SMRAD51 decreases homologous recombination without stimulating alternative nonconservative pathways and induces replication stress.SMRAD51 expression generates systemic inflammation in mice.SMRAD51 particularly affects highly proliferative tissues.SMRAD51 expression leads to premature aging without stimulating tumorigenesis.SMRAD51 impedes tumor formation and growth in a mouse model of breast cancer. [ABSTRACT FROM AUTHOR]

Highlights: What are the main findings? The job exposure matrix (JEM) provides annual means of PM10 concentrations for Parisian subway workers. Annual PM10 concentrations are estimated over the period of 2004–2020. What is the implication of the main finding? The JEM approach is relevant to assess occupational long-term exposure to subway PM10. The JEM will allow to examen the health effects of long-term exposure to subway PM10. Introduction: Health effects after long-term exposure to subway particulate matter (PM) remain unknown due to the lack of individual PM exposure data. This study aimed to apply the job exposure matrix (JEM) approach to retrospectively assess occupational exposure to PM in the Parisian subway. Methods: Job, the line and sector of the transport network, as well as calendar period were four JEM dimensions. For each combination of these dimensions, we generated statistical models to estimate the annual average PM10 concentration using data from an exhaustive inventory of the PM measurement campaigns conducted between 2004 and 2020 in the Parisian subway and historical data from the Parisian air pollution monitoring network. The resulting JEM and its exposure estimates were critically examined by experts using the uncertainty analysis framework. Results: The resulting JEM allows for the assignment of the estimated annual PM10 concentration to three types of professionals working in the subway: locomotive operators, station agents, and security guards. The estimates' precision and validity depend on the amount and quality of PM10 measurement data used in the job-, line-, and sector-specific models. Models using large amounts of personal exposure measurement data produced rather robust exposure estimates compared to models with lacunary data (i.e., in security guards). The analysis of uncertainty around the exposure estimates allows for the identification of the sources of uncertainty and parameters to be addressed in the future in order to refine and/or improve the JEM. Conclusions: The JEM approach seems relevant for the retrospective exposure assessment of subway workers. When applied to available data on PM10, it allows for the estimation of this exposure in locomotive operators and station agents with an acceptable validity. Conversely, for security guards, the current estimates have insufficient validity to recommend their use in an epidemiological study. Therefore, the current JEM should be considered as a valid prototype, which shall be further improved using more robust measurements for some jobs. This JEM can also be further refined by considering additional exposure determinants. [ABSTRACT FROM AUTHOR]

DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2‐7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de‐ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested.

Summary: Mammalian cells deficient in ATR or Chk1 display moderate replication fork slowing and increased initiation density, but the underlying mechanisms have remained unclear. We show that exogenous deoxyribonucleosides suppress both replication phenotypes in Chk1-deficient, but not ATR-deficient, cells. Thus, in the absence of exogenous stress, depletion of either protein impacts the replication dynamics through different mechanisms. In addition, Chk1 deficiency, but not ATR deficiency, triggers nuclease-dependent DNA damage. Avoiding damage formation through invalidation of Mus81-Eme2 and Mre11, or preventing damage signaling by turning off the ATM pathway, suppresses the replication phenotypes of Chk1-deficient cells. Damage and resulting DDR activation are therefore the cause, not the consequence, of replication dynamics modulation in these cells. Together, we identify moderate reduction of precursors available for replication as an additional outcome of DDR activation. We propose that resulting fork slowing, and subsequent firing of backup origins, helps replication to proceed along damaged templates. : Técher et al. show that modulation of DNA replication dynamics in Chk1-deficient mammalian cells is the consequence of DNA damage arising through unscheduled Mus81-Eme2 and Mre11 activation. Signaling of this damage by the ATM pathway impedes fork progression through dNTP shortage.

Over the last decade, CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have emerged as promising anticancer drugs. Numerous studies have demonstrated that CDK4/6 inhibitors efficiently block the pRb-E2F pathway and induce cell cycle arrest in pRb-proficient cells. Based on these studies, the inhibitors have been approved by the FDA for treatment of advanced hormonal receptor (HR) positive breast cancers in combination with hormonal therapy. However, some evidence has recently shown unexpected effects of the inhibitors, underlining a need to characterize the effects of CDK4/6 inhibitors beyond pRb. Our study demonstrates how palbociclib impairs origin firing in the DNA replication process in pRb-deficient cell lines. Strikingly, despite the absence of pRb, cells treated with palbociclib synthesize less DNA while showing no cell cycle arrest. Furthermore, this CDK4/6 inhibitor treatment disturbs the temporal program of DNA replication and reduces the density of replication forks. Cells treated with palbociclib show a defect in the loading of the Pre-initiation complex (Pre-IC) proteins on chromatin, indicating a reduced initiation of DNA replication. Our findings highlight hidden effects of palbociclib on the dynamics of DNA replication and of its cytotoxic consequences on cell viability in the absence of pRb. This study provides a potential therapeutic application of palbociclib in combination with other drugs to target genomic instability in pRB-deficient cancers. [ABSTRACT FROM AUTHOR]

Summary: Faithful DNA replication is essential for the maintenance of genome integrity. Incomplete genome replication leads to DNA breaks and chromosomal rearrangements, which are causal factors in cancer and other human diseases. Despite their importance, the molecular mechanisms that control human genome stability are incompletely understood. Here, we report a pathway that is required for human genome replication and stability. This pathway has three components: an E3 ubiquitin ligase, a transcriptional repressor, and a replication protein. The E3 ubiquitin ligase RBBP6 ubiquitinates and destabilizes the transcriptional repressor ZBTB38. This repressor negatively regulates transcription and levels of the MCM10 replication factor on chromatin. Cells lacking RBBP6 experience reduced replication fork progression and increased damage at common fragile sites due to ZBTB38 accumulation and MCM10 downregulation. Our results uncover a pathway that ensures genome-wide DNA replication and chromosomal stability. : DNA replication duplicates the genome at every cell cycle. Miotto et al. have now identified a molecular pathway that ensures proper replication of the mammalian genome. Common fragile sites are regions in the mammalian genome that are prone to loss when DNA replication is suboptimal. The new data show that common fragile sites are exquisitely sensitive to the activity of this RBBP6/ZBTB38/MCM10 axis, suggesting that deregulation of these factors may underlie genome instability and human disease.