UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2021

Gaetana Sessa, Åsa Ehlén, Catharina von Nicolai , Aura Carreira (2021 Jul 23)

Missense Variants of Uncertain Significance: A Powerful Genetic Tool for Function Discovery with Clinical Implications

CancersMissense Variants of Uncertain Significance: A Powerful Genetic Tool for Function Discovery with Clinical Implications : DOI : https://doi.org/10.3390/cancers13153719 En savoir plus
Résumé

The breast cancer susceptibility gene BRCA2 encodes a multifunctional protein required for the accurate repair of DNA double-strand breaks and replicative DNA lesions. In addition, BRCA2 exhibits emerging important roles in mitosis. As a result, mutations in BRCA2 may affect chromosomal integrity in multiple ways. However, many of the BRCA2 mutations found in breast cancer patients and their families are single amino acid substitutions, sometimes unique, and their relevance in cancer risk remains difficult to assess. In this review, we focus on three recent reports that investigated variants of uncertain significance (VUS) located in the N-terminal region of BRCA2. In this framework, we make the case for how the functional evaluation of VUS can be a powerful genetic tool not only for revealing novel aspects of BRCA2 function but also for re-evaluating cancer risk. We argue that other functions beyond homologous recombination deficiency or « BRCAness » may influence cancer risk. We hope our discussion will help the reader appreciate the potential of these functional studies in the prevention and diagnostics of inherited breast and ovarian cancer. Moreover, these novel aspects in BRCA2 function might help find new therapeutic strategies.

Keywords: breast cancer susceptibility; functional assays; variants of uncertain significance.

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Manon Julien 1 2, Rania Ghouil 1 2, Ambre Petitalot 3 4, Sandrine M Caputo 3 4, Aura Carreira 2 4 5, Sophie Zinn-Justin 1 2 (2021 Jul 20)

Intrinsic Disorder and Phosphorylation in BRCA2 Facilitate Tight Regulation of Multiple Conserved Binding Events

Intrinsic Disorder and Phosphorylation in BRCA2 Facilitate Tight Regulation of Multiple Conserved Binding Events : DOI : https://doi.org/10.3390/biom11071060 En savoir plus
Résumé

The maintenance of genome integrity in the cell is an essential process for the accurate transmission of the genetic material. BRCA2 participates in this process at several levels, including DNA repair by homologous recombination, protection of stalled replication forks, and cell division. These activities are regulated and coordinated via cell-cycle dependent modifications. Pathogenic variants in BRCA2 cause genome instability and are associated with breast and/or ovarian cancers. BRCA2 is a very large protein of 3418 amino acids. Most well-characterized variants causing a strong predisposition to cancer are mutated in the C-terminal 700 residues DNA binding domain of BRCA2. The rest of the BRCA2 protein is predicted to be disordered. Interactions involving intrinsically disordered regions (IDRs) remain difficult to identify both using bioinformatics tools and performing experimental assays. However, the lack of well-structured binding sites provides unique functional opportunities for BRCA2 to bind to a large set of partners in a tightly regulated manner. We here summarize the predictive and experimental arguments that support the presence of disorder in BRCA2. We describe how BRCA2 IDRs mediate self-assembly and binding to partners during DNA double-strand break repair, mitosis, and meiosis. We highlight how phosphorylation by DNA repair and cell-cycle kinases regulate these interactions. We finally discuss the impact of cancer-associated variants on the function of BRCA2 IDRs and more generally on genome stability and cancer risk.

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Jakub Muraszko, Karol Kramarz, Bilge Argunhan, Kentaro Ito, Gabriela Baranowska, Yumiko Kurokawa, Yasuto Murayama, Hideo Tsubouchi, Sarah Lambert, Hiroshi Iwasaki, Dorota Dziadkowiec (2021 Jun 22)

Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast.

Nucleic acids research : DOI : gkab511 En savoir plus
Résumé

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.

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Belen Gómez-González, Gaetana Sessa, Aura Carreira, Andrés Aguilera (2021 Apr 14)

A new interaction between BRCA2 and DDX5 promotes the repair of DNA breaks at transcribed chromatin.

Molecular & cellular oncology : 1910474 : DOI : 10.1080/23723556.2021.1910474 En savoir plus
Résumé

In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2-DDX5 interaction is impaired in cells expressing the missense variant found in breast cancer patients.

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Andrey Kleshnin, Léa Monet, Marina Plays, Hugo Vaysset, Claire Rougeulle, Stéphan Vagner (2021 Jan 6)

Amid darkness, light will prevail – a report on the 2020 annual SFC meeting on “Dark genome and Cancer”

Bulletin du cancer : DOI : S0007-4551(20)30510-5 En savoir plus
Résumé

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Karol Kramarz, Anissia Ait Saada, Sarah A E Lambert (2021 Jan 1)

The Analysis of Recombination-Dependent Processing of Blocked Replication Forks by Bidimensional Gel Electrophoresis.

Methods in molecular biology (Clifton, N.J.) : 365-381 : DOI : 10.1007/978-1-0716-0644-5_25 En savoir plus
Résumé

The perturbation of the DNA replication process is a threat to genome stability and is an underlying cause of cancer development and numerous human diseases. It has become central to understanding how stressed replication forks are processed to avoid their conversion into fragile and pathological DNA structures. The engineering of replication fork barriers (RFBs) to conditionally induce the arrest of a single replisome at a defined locus has made a tremendous impact in our understanding of replication fork processing. Applying the bidimensional gel electrophoresis (2DGE) technique to those site-specific RFBs allows the visualization of replication intermediates formed in response to replication fork arrest to investigate the mechanisms ensuring replication fork integrity. Here, we describe the 2DGE technique applied to the site-specific RTS1-RFB in Schizosaccharomyces pombe and explain how this approach allows the detection of arrested forks undergoing nascent strands resection.

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(2021 Jan 1)

Monitoring Homologous Recombination Activity in Human Cells

.Methods Mol Biol : DOI : 10.1007/978-1-0716-0644-5_9 En savoir plus
Résumé

DNA double-strand breaks (DSBs) are among the most toxic lesions. This type of DNA damage is repaired by two major pathways, homologous recombination (HR), operating only in S/G2 cell-cycle phases and nonhomologous end joining (NHEJ) which is operative throughout the cell cycle. Because HR is a template-directed repair, it is generally less prone to errors and/or translocations than NHEJ.The HR pathway involves several effector proteins and regulators that modulate the efficiency of repair and limit the repair outside S/G2 phase. Some of the genes coding for these proteins are frequently mutated in human diseases such as cancer, and pathogenic mutations or variants identified in patients often alter the HR proficiency of the cells.This chapter describes a cell-based gene-targeting reporter assay in human cells to evaluate the repair of a site-specific DSB by HR . In it, a promoter-less fluorescent protein is encoded in a plasmid flanked by two homology arms directed to a safe-harbour locus in the genome. The expression of the fluorescent protein is driven by the promoter of the endogenous locus enabling to quantify the efficiency of HR by flow cytometry. This approach can be used to determine the requirement of certain proteins, protein domains, or protein modifications for HR . It can also be used to functionally evaluate variants of the genes encoding these proteins such as BRCA1, BRCA2, RAD51C, and PALB2; which may help assess their pathogenicity. Here, we use the homologous recombination mediator BRCA2 to illustrate the assay.

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Année de publication : 2020

(2020 Oct 1)

Flavaglines as natural products targeting eIF4A and prohibitins: From traditional Chinese medicine to antiviral activity against coronaviruses

Eur J Med Chem. : DOI : 10.1016/j.ejmech.2020.112653 En savoir plus
Résumé

Flavaglines are cyclopenta[b]benzofurans found in plants of the genus Aglaia, several species of which are used in traditional Chinese medicine. These compounds target the initiation factor of translation eIF4A and the scaffold proteins prohibitins-1 and 2 (PHB1/2) to exert various pharmacological activities, including antiviral effects against several types of viruses, including coronaviruses. This review is focused on the antiviral effects of flavaglines and their therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

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(2020 Aug 26)

Identification and Analysis of Different Types of UFBs

Methods Mol Biol. : DOI : 10.1007/978-1-0716-0644-5_13 En savoir plus
Résumé

Ultrafine anaphase bridges (UFBs) result from a defect in sister chromatid segregation during anaphase. They arise from particular DNA structures, mostly generated at specific loci in the human genome, such as centromeres, common fragile sites, telomeres, or ribosomal DNA. Increases in UFB frequency are a marker of genetic instability, and their detection has become a classic way of detecting such genetic instability over the last decade. Here we describe a protocol to stain different types of UFBs in adherent human cells.

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Sandra Cunha Silveira, Géraldine Buhagiar‑Labarchède, Rosine Onclercq‑Delic, Simon Gemble, Elias Bou Samra, Hamza Mameri, Patricia Duchambon, Christelle Machon, Jérôme Guitton & Mounira Amor‑Guéret (2020 Aug 17)

A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD+

Scientific Reports : 10 : 13907 : DOI : 10.1038/s41598-020-70874-6 En savoir plus
Résumé

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.

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Simon D Brown, Charlotte Audoynaud, Alexander Lorenz (2020 Jun 28)

Intragenic meiotic recombination in Schizosaccharomyces pombe is sensitive to environmental temperature changes.

Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology : 195-207 : DOI : 10.1007/s10577-020-09632-3 En savoir plus
Résumé

Changes in environmental temperature influence cellular processes and their dynamics, and thus affect the life cycle of organisms that are unable to control their cell/body temperature. Meiotic recombination is the cellular process essential for producing healthy haploid gametes by providing physical links (chiasmata) between homologous chromosomes to guide their accurate segregation. Additionally, meiotic recombination-initiated by programmed DNA double-strand breaks (DSBs)-can generate genetic diversity and, therefore, is a driving force of evolution. Environmental temperature influencing meiotic recombination outcome thus may be a crucial determinant of reproductive success and genetic diversity. Indeed, meiotic recombination frequency in fungi, plants and invertebrates changes with temperature. In most organisms, these temperature-induced changes in meiotic recombination seem to be mediated through the meiosis-specific chromosome axis organization, the synaptonemal complex in particular. The fission yeast Schizosaccharomyces pombe does not possess a synaptonemal complex. Thus, we tested how environmental temperature modulates meiotic recombination frequency in the absence of a fully-fledged synaptonemal complex. We show that intragenic recombination (gene conversion) positively correlates with temperature within a certain range, especially at meiotic recombination hotspots. In contrast, crossover recombination, which manifests itself as chiasmata, is less affected. Based on our observations, we suggest that, in addition to changes in DSB frequency, DSB processing could be another temperature-sensitive step causing temperature-induced recombination rate alterations.

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(2020 Jun 1)

Preclinical Efficacy of Humanized, non-FcgammaR Binding Anti-CD3 Antibodies in T-cell Acute Lymphoblastic Leukemia

Blood. : DOI : 10.1182/blood.2019003801 En savoir plus
Résumé

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for about 20% ALL cases. Intensive chemotherapy regimens result in >85% cure rates in children and <50% in adults, calling for the search of novel therapeutic strategies. While immune-based therapies have tremendously improved the treatment of B-ALL and other B-cell malignancies, they are not available yet in T-ALL. We report here that humanized, non-FcgR binding monoclonal antibodies to CD3 have anti-leukemic properties in xenograft (PDX) models of CD3+ T-ALL, resulting in prolonged host survival. We also report that these antibodies cooperate with chemotherapy to enhance anti-leukemic effects and host survival. As these antibodies show only minor, manageable side effects in humans, they offer a new therapeutic option for the treatment of T-ALL. Our results also show that the anti-leukemic properties of anti-CD3 mAbs are largely independent Fcg-receptor mediated pathways in T-ALL PDX.

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Simon Gemble, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Gaëlle Fontaine, Sarah Lambert, Mounira Amor-Guéret (2020 May 14)

Topoisomerase IIα prevents ultrafine anaphase bridges by two mechanisms.

Open biology : 190259 : DOI : 10.1098/rsob.190259 En savoir plus
Résumé

Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase-anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.

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Åsa Ehlén, Charlotte Martin, Simona Miron, Manon Julien, François-Xavier Theillet, Virginie Ropars, Gaetana Sessa, Romane Beaurepere, Virginie Boucherit, Patricia Duchambon, Ahmed El Marjou, Sophie Zinn-Justin, Aura Carreira (2020 Apr 14)

Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1.

Nature communications : 1819 : DOI : 10.1038/s41467-020-15689-9 En savoir plus
Résumé

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.

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Samah Matmati, Sarah Lambert, Vincent Géli, Stéphane Coulon (2020 Mar 12)

Telomerase Repairs Collapsed Replication Forks at Telomeres.

Cell reports : 3312-3322.e3 : DOI : S2211-1247(20)30233-3 En savoir plus
Résumé

Telomeres are difficult-to-replicate sites whereby replication itself may threaten telomere integrity. We investigate, in fission yeast, telomere replication dynamics in telomerase-negative cells to unmask problems associated with telomere replication. Two-dimensional gel analysis reveals that replication of telomeres is severely impaired and correlates with an accumulation of replication intermediates that arises from stalled and collapsed forks. In the absence of telomerase, Rad51, Mre11-Rad50-Nbs1 (MRN) complex, and its co-factor CtIP become critical to maintain telomeres, indicating that homologous recombination processes these intermediates to facilitate fork restart. We further show that a catalytically dead mutant of telomerase prevents Ku recruitment to telomeres, suggesting that telomerase and Ku both compete for the binding of telomeric-free DNA ends that are likely to originate from a reversed fork. We infer that Ku removal at collapsed telomeric forks allows telomerase to repair broken telomeres, thereby shielding telomeres from homologous recombination.

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