Biology subjects

Ribeyre, C.

Publications and source records attributed to Ribeyre, C..

9 recordsLinked to original sources

bioRxiv2025

Condensin and topoisomerases cooperate to relieve topological stress at stalled replication forks

Resolving complex topological structures at replication forks is vital for successful DNA replication, but the mechanisms are little understood. Evidence from diverse eukaryotes suggests that condensin - which promotes chromosome condensation in M phase - might also act during S phase to facilitate relaxation of torsional stress by topoisomerases. Here, we show in yeast and human cells that condensin binds stressed replication forks, where it cooperates with topoisomerases I and II to promote resection of the nascent DNA and restart replication. Our findings suggest that condensin acts with topoisomerase I at reversed forks to convert positively supercoiled DNA into structures that are subsequently relaxed by topoisomerase 2, allowing the fork to resume replication. These findings uncover an important, evolutionarily conserved role for condensin in handling topological constraints at arrested forks that is reminiscent of its function in chromosome segregation and might prevent formation of toxic chromosome structures during fork arrest and reversal.

genetics↗

bioRxiv2023

Cell-cycle dependent inhibition of BRCA1 signaling by the lysine methyltransferase SET8

The cell-cycle regulated methyltransferase SET8 is the sole enzyme responsible for the mono-methylation of histone H4 at lysine 20 (H4K20) that is the substrate for di- and tri-methylation mainly by SUV4-20Hs enzymes. Both SET8 and SUV4-20Hs have been implicated in regulating DNA repair pathway choice through the inverse affinities of BRCA1-BARD1 and 53BP1 complexes for disparate methylation states of H4K20. However, the precise and respective functions of each H4K20 methyltransferases in DNA repair pathways remained to be clarified. Here, we show that SET8 acts as a potent chromatin inhibitor of homologous recombination and that its timely degradation during DNA replication is essential for the spontaneous nuclear focal accumulation of BRCA1 and RAD51 complexes during S-phase. Strikingly, the anti-recombinogenic function of SET8 is independent of SUV4-20H activity but requires the subsequent recruitment of the ubiquitin ligase RNF168. Moreover, we show that SET8-induced BRCA1 inhibition is not necessarily related to the loss of BARD1 binding to unmethylated histone H4K20. Instead, it is largely caused by the accumulation of 53BP1 in a manner depending on the concerted activities of SET8 and RNF168 on chromatin. Conversely, the lack of SET8 and H4K20 mono-methylation on newly assembly chromatin after DNA replication led to the untimely accumulation of BRCA1 on chromatin at the subsequent G1 phase. Altogether, these results establish the de novo activity of SET8 on chromatin as a primordial epigenetic lock of BRCA1-mediated HR pathway during the cell cycle.

molecular biology↗

bioRxiv2023

Replication poison treated BRCA1-deficient breast cancers are prone to MRE11 over-resection resulting in single strand DNA accumulation and mitotic catastrophe

BRCA1, BRCA2 and RAD51, key players of homologous recombination (HR) repair, are also involved in stalled DNA replication fork protection and repair. BRCA1-deficiency is encountered in 25% of Triple Negative Breast Cancer (TNBC). Here we investigated the sensitivity of BRCA1-deficient TNBC cell models to gemcitabine a frequently used replication poison that does not alter DNA structure. We show that BRCA1-deficient models, in contrast to their isogenic BRCA1-proficient counterparts, are superiorly sensitive to gemcitabine, accumulate massive levels of single strand DNA (ssDNA), in absence of RPA and RAD51 signals and elevated double strand break (DSB) numbers leading to cell death. Remarkably, ssDNA accumulation in gemcitabine-treated BRCA1-deficient cells was strongly diminished by the MRE11 inhibitor mirin, while it did not affect ssDNA levels resulting from PARP inhibitor olaparib treatment. The central role of MRE11 DNA resection strongly suggested that replication fork reversal may be important in response to replication poisoning by gemcitabine in BRCA1-deficient models. Furthermore, we demonstrate that gemcitabine-treated BRCA1-deficient cells showing massive ssDNA accumulation slipped into mitosis and produced mitotic bridges and micronuclei (MN) showing strong BrdU and {gamma}H2AX staining. Noticeably these BrdU-positive MN and DNA bridges triggered cGAS sensing. Our data, thus, strongly suggest that gemcitabine treatment could be beneficial in BRCA1-deficient TNBC both in terms of cancer cell death, but possibly as well in terms of antitumor immune response.

cancer biology↗

bioRxiv2022

GNL3 regulates replication origin firing and protects stalled replication forks

DNA replication requires specific proteins that protect replication forks and so prevent the formation of DNA lesions that may damage the genome. Identification of new proteins involved in these processes is essential to understand how cancer cells tolerate DNA lesions. Here we show that human GNL3/nucleostemin, a GTP-binding protein localized mostly in the nucleolus and highly expressed in cancer cells, prevents nuclease-dependent resection of nascent DNA in response to exogenous replication stress. We demonstrate that inhibition of origin firing decreases this resection, indicating that the increased replication origin firing seen upon GNL3 depletion mainly accounts for the observed DNA resection. We show that GNL3 and DNA replication initiation factor ORC2 interact in the nucleolus and that the concentration of GNL3 in the nucleolus is required to limit DNA resection in response to replicative stress. We propose that the accurate control of origin firing by GNL3, possibly through the regulation of ORC2 sub-nuclear localization, is critical to prevent nascent DNA resection in response to replication stress.

molecular biology↗

bioRxiv2021

Cytidine deaminase protects pancreatic cancer cells from replicative stress and drives resistance to DNA-targeting drugs

Chronic DNA replication stress and genome instability are two hallmarks of cancer that fuel oncogenesis and tumor diversity. Therapeutic approaches aimed to leverage tumor-specific replication stress to intolerable levels or to expose vulnerabilities for synthetic lethality purposes have recently gained momentum, especially for pancreatic cancer, a disease with no cure. However, the current knowledge regarding the molecular mechanisms involved in the replication stress response in pancreatic tumors is limited. Cytidine deaminase (CDA) is involved in the pyrimidine salvage pathway for DNA and RNA synthesis. Loss of CDA induces genomic instability in Bloom Syndrome, and CDA protects tumor cells from chemotherapy with pyrimidine analogs. Here, we show that CDA is overexpressed in genetically unstable pancreatic tumors, associates with a DNA replication signature, and is instrumental for experimental tumor growth. In cancer cells, CDA promotes DNA replication, increases replication fork speed, and controls replication stress and genomic stability levels. CDA expression is predictive of DNA-damaging drug efficacy and targeting CDA relieves resistance to chemotherapy in patients models, both in vitro and in vivo. Our findings shed new light on the mechanisms by which pancreatic cancer cells control replication stress, and highlight targeting of CDA as a potential therapeutic strategy to defeat tumor resistance to treatment.

cancer biology↗

bioRxiv2019

KDM5 histone-demethylases contribute to replication stress response and tolerance.

KDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of Ribonucleotide Reductase. Second, they are required for optimal activation of Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. This role in Chk1 activation is probably direct since KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU, in a manner that is independent of their demethylase activity. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression.

cancer biology↗

bioRxiv2019

Experimental system and image analysis software for high throughput phenotyping of mycorrhizal growth response in Brachypodium distachyon

O_LIPlant growth response to Arbuscular Mycorrhizal (AM) fungi is variable and depends on genetic and environment factors that still remain largely unknown. Identification of these factors can be envisaged using high-throughput and accurate plant phenotyping.\nC_LIO_LIWe setup experimental conditions based on a two-compartment system allowing to measure Brachypodium distachyon mycorhizal growth response (MGR) in an automated phenotyping greenhouse. We developed a new image analysis software \"IPSO Phen\" to estimate of B. distachyon aboveground biomass.\nC_LIO_LIWe found a positive MGR in the B. distachyon Bd3-1 genotype inoculated with the AM fungi Rhizophagus irregularis only if nitrogen and phosphorus were added together in the compartment restricted to AM fungi. Using this condition, we found genetic diversity in B. distachyon for MGR ranging from positive to negative MGR depending on the plant genotype tested.\nC_LIO_LIOur result on the interaction between nitrogen and phosphorus for MGR in B. distachyon opens new perspectives about AM functioning. In addition, our open-source software allowing to test and run image analysis parameters on large amount of images generated by automated plant phenotyping facilities, will help to screen large panels of genotypes and environmental conditions to identify the factors controlling the MGR.\nC_LI

plant biology↗

bioRxiv2019

RIF1 ensures the organization of replication factories upon DNA replicative stress.

Eukaryotic genomes are duplicated from thousands of replication origins that fire sequentially forming a defined spatiotemporal pattern of replication clusters. The temporal order of DNA replication is determined by chromatin architecture and, more specifically, by chromatin contacts that are stabilized by RIF1. Here we show that RIF1 localizes in close proximity to newly synthesized DNA. In cells exposed to the DNA replication inhibitor aphidicolin, suppression of RIF1 markedly decreased the efficacy of protein isolation on nascent DNA (iPOND), suggesting that the iPOND procedure is biased by chromatin topology. RIF1 was required to limit the accumulation of DNA lesions induced by aphidicolin treatment and promoted the recruitment of cohesins in the vicinity of nascent DNA. Collectively, the data suggest that the stabilization of chromatin topology by RIF1 limits replication-associated genomic instability.

cell biology↗

bioRxiv2018

Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites.

Metabolic alterations support cellular transformation. Noticeably, cellular transformation and cancer progression is associated with accumulation of dihydrouracil and dihydrothymine. However, it remains elusive how dihydropyrimidine metabolites affect cellular phenotypes. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP). This zinc metalloenzyme is upregulated in solid tumors but not in the corresponding normal tissues. Here we show that DHP silencing in transformed cell lines is cytotoxic. Increasing the level of dihydropyrimidines inhibited DNA replication and transcription. It induced replication stress signaling by ATR and p53 stabilization. Remarkably, cells lacking DHP accumulated DNA-protein crosslinks (DPCs). Among DPCs, covalently trapped translesion DNA polymerase {eta} contributed to the cytotoxicity of dihydropyrimidines. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits the activity of recombinant human DHP. Cellular treatment with dihydromyricetin triggered DPCs-dependent DNA replication stress. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.

cancer biology↗