Recombinaison, Réplication et Stabilité du génome

Publications de l’équipe

Année de publication : 2021

Antony Carr, Sarah Lambert (2021 Aug 4)

Recombination-dependent replication: new perspectives from site-specific fork barriers.

Current opinion in genetics & development : 129-135 : DOI : S0959-437X(21)00094-0 En savoir plus
Résumé

Replication stress (RS) is intrinsic to normal cell growth, is enhanced by exogenous factors and elevated in many cancer cells due to oncogene expression. Most genetic changes are a result of RS and the mechanisms by which cells tolerate RS has received considerable attention because of the link to cancer evolution and opportunities for cancer cell-specific therapeutic intervention. Site-specific replication fork barriers have provided unique insights into how cells respond to RS and their recent use has allowed a deeper understanding of the mechanistic and spatial mechanism that restart arrested forks and how these correlate with RS-dependent mutagenesis. Here we review recent data from site-specific fork arrest systems used in yeast and highlight their strengths and limitations.

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Charlotte Audoynaud, Stéphan Vagner, Sarah Lambert (2021 Jul 5)

Non-homologous end-joining at challenged replication forks: an RNA connection?

Trends in genetics : TIG : DOI : S0168-9525(21)00166-9 En savoir plus
Résumé

Defective DNA replication, known as ‘replication stress’, is a source of DNA damage, a hallmark of numerous human diseases, including cancer, developmental defect, neurological disorders, and premature aging. Recent work indicates that non-homologous end-joining (NHEJ) is unexpectedly active during DNA replication to repair replication-born DNA lesions and to safeguard replication fork integrity. However, erroneous NHEJ events are deleterious to genome stability. RNAs are novel regulators of NHEJ activity through their ability to modulate the assembly of repair complexes in trans. At DNA damage sites, RNAs and DNA-embedded ribonucleotides modulate repair efficiency and fidelity. We discuss here how RNAs and associated proteins, including RNA binding proteins, may regulate NHEJ to sustain genome stability during DNA replication.

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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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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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Année de publication : 2020

Karol Kramarz, Kamila Schirmeisen, Virginie Boucherit, Anissia Ait Saada, Claire Lovo, Benoit Palancade, Catherine Freudenreich, Sarah A E Lambert (2020 Nov 7)

The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal.

Nature communications : 5643 : DOI : 10.1038/s41467-020-19516-z En savoir plus
Résumé

Nuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR). Anchorage to NPCs allows SUMO removal by the SENP SUMO protease Ulp1 and the proteasome, promoting timely resumption of DNA synthesis. Preventing Pli1-mediated SUMO chains was sufficient to bypass the need for anchorage to NPCs and the inhibitory effect of poly-SUMOylation on HR-mediated DNA synthesis. Our work establishes a novel spatial control of Recombination-Dependent Replication (RDR) at a unique sequence that is distinct from mechanisms engaged at collapsed-forks and breaks within repeated sequences.

https://www-nature-com.insb.bib.cnrs.fr/articles/s41467-020-19516-z

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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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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
Résumé

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.

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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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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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Anna Barg-Wojas, Jakub Muraszko, Karol Kramarz, Kamila Schirmeisen, Gabriela Baranowska, Antony M Carr, Dorota Dziadkowiec (2020 Feb 10)

DNA translocases Rrp1 and Rrp2 have distinct roles at centromeres and telomeres that ensure genome stability.

Journal of cell science : DOI : jcs230193 En savoir plus
Résumé

The regulation of telomere and centromere structure and function is essential for maintaining genome integrity. Rrp1 and Rrp2 are orthologues of Uls1, a SWI2/SNF2 DNA translocase and SUMO-targeted ubiquitin ligase. Here, we show that Rrp1 or Rrp2 overproduction leads to chromosome instability and growth defects, a reduction in global histone levels and mislocalisation of centromere-specific histone Cnp1. These phenotypes depend on putative DNA translocase activities of Rrp1 and Rrp2, suggesting that Rrp1 and Rrp2 may be involved in modulating nucleosome dynamics. Furthermore, we confirm that Rrp2, but not Rrp1, acts at telomeres, reflecting a previously described interaction between Rrp2 and Top2. In conclusion, we identify roles for Rrp1 and Rrp2 in maintaining centromere function by modulating histone dynamics, contributing to the preservation of genome stability during vegetative cell growth.

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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
Résumé

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.

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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
Résumé

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.

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Sarah Lambert (2019 Mar 3)

Unstable genomes promote inflammation.

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

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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
Résumé

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.

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