DNA Recombination, Replication and Genome Stability

Team Publications

Year of 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 Learn more

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.


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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 Learn more

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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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 Learn more

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 Learn more

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 Learn more

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 Learn more

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 Learn more

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 Learn more

Year of 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 Learn more

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 Learn more

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 Learn more

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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 Learn more

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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Year of 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 Learn more

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.

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Year of 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 Learn more

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.

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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 Learn more

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.

Free acces : authors.elsevier.com/a/1U~li3vVUP2C0m

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