Recombinaison de l’ ADN, réplication et stabilité du génome

Publications de l’équipe

Année de publication : 2020

Karol Kramarz, Anissia Ait Saada, Sarah A E Lambert (2020 Aug 26)

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

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.

L Boeckemeier, R Kraehenbuehl, A Keszthelyi, M U Gasasira, E G Vernon, R Beardmore, C B Vågbø, D Chaplin, S Gollins, H E Krokan, S A E Lambert, B Paizs, E Hartsuiker (2020 May 29)

Mre11 exonuclease activity removes the chain-terminating nucleoside analog gemcitabine from the nascent strand during DNA replication.

Science advances : eaaz4126 : DOI : 10.1126/sciadv.aaz4126 En savoir plus

The Mre11 nuclease is involved in early responses to DNA damage, often mediated by its role in DNA end processing. mutations and aberrant expression are associated with carcinogenesis and cancer treatment outcomes. While, in recent years, progress has been made in understanding the role of Mre11 nuclease activities in DNA double-strand break repair, their role during replication has remained elusive. The nucleoside analog gemcitabine, widely used in cancer therapy, acts as a replication chain terminator; for a cell to survive treatment, gemcitabine needs to be removed from replicating DNA. Activities responsible for this removal have, so far, not been identified. We show that Mre11 3′ to 5′ exonuclease activity removes gemcitabine from nascent DNA during replication. This contributes to replication progression and gemcitabine resistance. We thus uncovered a replication-supporting role for Mre11 exonuclease activity, which is distinct from its previously reported detrimental role in uncontrolled resection in recombination-deficient cells.

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

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.

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

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.

Lambert, S. Borde, V. Charbonnier, J. B. Dantzer, F. Espeli, O. Guirouilh-Barbat, J. Llorente, B. Legube, G. Prioleau, M. N. Radicella, P. (2020 Feb 1)

Des mécanismes moléculaires aux applications cliniques. L’essentiel du Colloque Réplication-Réparation-Recombinaison 2019

Bull Cancer : 283-287 : DOI : 10.1016/j.bulcan.2020.01.003 En savoir plus

Année de publication : 2019

Julien Hardy, Dingli Dai, Anissia Ait Saada, Ana Teixeira-Silva, Louise Dupoiron, Fatemeh Mojallali, Karine Fréon, Francoise Ochsenbein, Brigitte Hartmann, Sarah Lambert (2019 Oct 4)

Histone deposition promotes recombination-dependent replication at arrested forks.

PLoS genetics : e1008441 : DOI : 10.1371/journal.pgen.1008441 En savoir plus

Replication stress poses a serious threat to genome stability. Recombination-Dependent-Replication (RDR) promotes DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways after DNA repair, crosstalk coupling RDR and chromatin assembly is largely unexplored. The fission yeast Chromatin Assembly Factor-1, CAF-1, is known to promote RDR. Here, we addressed the contribution of histone deposition to RDR. We expressed a mutated histone, H3-H113D, to genetically alter replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition, by CAF-1 and Asf1, promotes RDR by preventing Rqh1-mediated disassembly of joint-molecules. The recombination factor Rad52 promotes CAF-1 binding to sites of recombination-dependent DNA synthesis, indicating that histone deposition occurs downstream Rad52. Histone deposition and Rqh1 activity act synergistically to promote cell resistance to camptothecin, a topoisomerase I inhibitor that induces replication stress. Moreover, histone deposition favors non conservative recombination events occurring spontaneously in the absence of Rqh1, indicating that the stabilization of joint-molecules by histone deposition also occurs independently of Rqh1 activity. These results indicate that histone deposition plays an active role in promoting RDR, a benefit counterbalanced by stabilizing at-risk joint-molecules for genome stability.

Anissia Ait-Saada, Olga Khorosjutina, Jiang Chen, Karol Kramarz, Vladimir Maksimov, J Peter Svensson, Sarah Lambert, Karl Ekwall (2019 Oct 1)

Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks.

Life science alliance : DOI : e201900433 En savoir plus

Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break. Furthermore, we use an inducible replication fork barrier system to show that Fft3 has two distinct roles at blocked DNA replication forks. First, Fft3 is needed for the resection of nascent strands, and second, it is required to restart the blocked forks. The latter function is independent of its ATPase activity.

Sarah Lambert (2019 Mar 3)

Unstable genomes promote inflammation.

Nature : 41-42 : DOI : 10.1038/d41586-019-00510-5 En savoir plus

Hannah L Klein, Giedrė Bačinskaja, Jun Che, Anais Cheblal, Rajula Elango, Anastasiya Epshtein, Devon M Fitzgerald, Belén Gómez-González, Sharik R Khan, Sandeep Kumar, Bryan A Leland, Léa Marie, Qian Mei, Judith Miné-Hattab, Alicja Piotrowska, Erica J Polleys, Christopher D Putnam, Elina A Radchenko, Anissia Ait Saada, Cynthia J Sakofsky, Eun Yong Shim, Mathew Stracy, Jun Xia, Zhenxin Yan, Yi Yin, Andrés Aguilera, Juan Lucas Argueso, Catherine H Freudenreich, Susan M Gasser, Dmitry A Gordenin, James E Haber, Grzegorz Ira, Sue Jinks-Robertson, Megan C King, Richard D Kolodner, Andrei Kuzminov, Sarah Ae Lambert, Sang Eun Lee, Kyle M Miller, Sergei M Mirkin, Thomas D Petes, Susan M Rosenberg, Rodney Rothstein, Lorraine S Symington, Pawel Zawadzki, Nayun Kim, Michael Lisby, Anna Malkova (2019 Jan 7)

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Microbial cell (Graz, Austria) : 1-64 : DOI : 10.15698/mic2019.01.664 En savoir plus

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.


Année de publication : 2018

Anissia Ait Saada, Sarah A E Lambert, Antony M Carr (2018 Aug 25)

Preserving replication fork integrity and competence via the homologous recombination pathway.

DNA repair : DOI : S1568-7864(18)30182-4 En savoir plus

Flaws in the DNA replication process have emerged as a leading driver of genome instability in human diseases. Alteration to replication fork progression is a defining feature of replication stress and the consequent failure to maintain fork integrity and complete genome duplication within a single round of S-phase compromises genetic integrity. This includes increased mutation rates, small and large scale genomic rearrangement and deleterious consequences for the subsequent mitosis that result in the transmission of additional DNA damage to the daughter cells. Therefore, preserving fork integrity and replication competence is an important aspect of how cells respond to replication stress and avoid genetic change. Homologous recombination is a pivotal pathway in the maintenance of genome integrity in the face of replication stress. Here we review our recent understanding of the mechanisms by which homologous recombination acts to protect, restart and repair replication forks. We discuss the dynamics of these genetically distinct functions and their contribution to faithful mitoticsegregation.


Année de publication : 2017

Ana Teixeira-Silva, Anissia Ait Saada, Julien Hardy, Ismail Iraqui, Marina Charlotte Nocente, Karine Fréon, Sarah A E Lambert (2017 Dec 7)

The end-joining factor Ku acts in the end-resection of double strand break-free arrested replication forks.

Nature communications : 1982 : DOI : 10.1038/s41467-017-02144-5 En savoir plus

Replication requires homologous recombination (HR) to stabilize and restart terminally arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily double strand breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe. Here, we report that fork-resection is a two-step process regulated by the non-homologous end joining factor Ku. An initial resection mediated by MRN-Ctp1 removes Ku from terminally arrested forks, generating ~110 bp sized gaps obligatory for subsequent Exo1-mediated long-range resection and replication restart. The mere lack of Ku impacts the processing of arrested forks, leading to an extensive resection, a reduced recruitment of RPA and Rad51 and a slower fork-restart process. We propose that terminally arrested forks undergo fork reversal, providing a single DNA end for Ku binding. We uncover a role for Ku in regulating end-resection of unbroken forks and in fine-tuning HR-mediated replication restart.

Anissia Ait Saada, Ana Teixeira-Silva, Ismail Iraqui, Audrey Costes, Julien Hardy, Giulia Paoletti, Karine Fréon, Sarah A E Lambert (2017 May 4)

Unprotected Replication Forks Are Converted into Mitotic Sister Chromatid Bridges.

Molecular cell : 398-410.e4 : DOI : 10.1016/j.molcel.2017.04.002 En savoir plus

Replication stress and mitotic abnormalities are key features of cancer cells. Temporarily paused forks are stabilized by the intra-S phase checkpoint and protected by the association of Rad51, which prevents Mre11-dependent resection. However, if a fork becomes dysfunctional and cannot resume, this terminally arrested fork is rescued by a converging fork to avoid unreplicated parental DNA during mitosis. Alternatively, dysfunctional forks are restarted by homologous recombination. Using fission yeast, we report that Rad52 and the DNA binding activity of Rad51, but not its strand-exchange activity, act to protect terminally arrested forks from unrestrained Exo1-nucleolytic activity. In the absence of recombination proteins, large ssDNA gaps, up to 3 kb long, occur behind terminally arrested forks, preventing efficient fork merging and leading to mitotic sister chromatid bridging. Thus, Rad52 and Rad51 prevent temporarily and terminally arrested forks from degrading and, despite the availability of converging forks, converting to anaphase bridges causing aneuploidy and cell death.

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Année de publication : 2016

Simon Gemble, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Denis Biard, Sarah Lambert, Mounira Amor-Guéret (2016 Aug 15)

A balanced pyrimidine pool is required for optimal Chk1 activation to prevent ultrafine anaphase bridge formation.

Journal of cell science : 3167-77 : DOI : 10.1242/jcs.187781 En savoir plus

Cytidine deaminase (CDA) deficiency induces an excess of cellular dCTP, which reduces basal PARP-1 activity, thereby compromising complete DNA replication, leading to ultrafine anaphase bridge (UFB) formation. CDA dysfunction has pathological implications, notably in cancer and in Bloom syndrome. It remains unknown how reduced levels of PARP-1 activity and pyrimidine pool imbalance lead to the accumulation of unreplicated DNA during mitosis. We report that a decrease in PARP-1 activity in CDA-deficient cells impairs DNA-damage-induced Chk1 activation, and, thus, the downstream checkpoints. Chemical inhibition of the ATR-Chk1 pathway leads to UFB accumulation, and we found that this pathway was compromised in CDA-deficient cells. Our data demonstrate that ATR-Chk1 acts downstream from PARP-1, preventing the accumulation of unreplicated DNA in mitosis, and, thus, UFB formation. Finally, delaying entry into mitosis is sufficient to prevent UFB formation in both CDA-deficient and CDA-proficient cells, suggesting that both physiological and pathological UFBs are derived from unreplicated DNA. Our findings demonstrate an unsuspected requirement for a balanced nucleotide pool for optimal Chk1 activation both in unchallenged cells and in response to genotoxic stress.


Année de publication : 2015

Simon Gemble, Akshay Ahuja, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Julien Dairou, Denis S F Biard, Sarah Lambert, Massimo Lopes, Mounira Amor-Guéret (2015 Jul 16)

Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

PLoS genetics : e1005384 : DOI : 10.1371/journal.pgen.1005384 En savoir plus

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at « difficult-to-replicate » sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3′-5′ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

Saed Mohebi, Sarah A E Lambert, Antony M Carr (2015 Apr 29)

Analyzing the Response to Dysfunction Replication Forks Using the RTS1 Barrier System in Fission Yeast.

Methods in molecular biology (Clifton, N.J.) : 239-59 : DOI : 10.1007/978-1-4939-2596-4_15 En savoir plus

The study of how eukaryotic cells overcome problems associated with dysfunctional DNA replication forks is assisted by experimental systems that allow site-specific replication fork arrest. Here we provide protocols for the use of the fission yeast RTS1 replication fork barrier. The RTS1 barrier is a directional, or polar, replication fork barrier that evolved to ensure directional replication of the fission yeast mating-type locus. We have moved the 859 bp RTS1 sequence to another locus in the genome and demonstrated that it arrests replication forks in a dysfunctional confirmation and that replication is restarted within ~20 min by the action of homologous recombination. We describe here the barrier constructs currently available, the methods by which we regulate the activity of the barrier, how to synchronize cells for analysis of replication intermediates by 2D gel electrophoresis, and the use of a replication slippage assay to measure fork fidelity.