Dynamique des chromosomes et recombinaison

Publications de l’équipe

Année de publication : 2015

Hardeep Kaur, Arnaud De Muyt, Michael Lichten (2015 Feb 21)

Top3-Rmi1 DNA single-strand decatenase is integral to the formation and resolution of meiotic recombination intermediates.

Molecular cell : 583-94 : DOI : 10.1016/j.molcel.2015.01.020 En savoir plus
Résumé

The topoisomerase III (Top3)-Rmi1 heterodimer, which catalyzes DNA single-strand passage, forms a conserved complex with the Bloom’s helicase (BLM, Sgs1 in budding yeast). This complex has been proposed to regulate recombination by disassembling double Holliday junctions in a process called dissolution. Top3-Rmi1 has been suggested to act at the end of this process, resolving hemicatenanes produced by earlier BLM/Sgs1 activity. We show here that, to the contrary, Top3-Rmi1 acts in all meiotic recombination functions previously associated with Sgs1, most notably as an early recombination intermediate chaperone, promoting regulated crossover and noncrossover recombination and preventing aberrant recombination intermediate accumulation. In addition, we show that Top3-Rmi1 has important Sgs1-independent functions that ensure complete recombination intermediate resolution and chromosome segregation. These findings indicate that Top3-Rmi1 activity is important throughout recombination to resolve strand crossings that would otherwise impede progression through both early steps of pathway choice and late steps of intermediate resolution.

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Année de publication : 2014

Valérie Borde, Michael Lichten (2014 Aug 16)

A timeless but timely connection between replication and recombination.

Cell : 697-8 : DOI : 10.1016/j.cell.2014.07.029 En savoir plus
Résumé

Initiation of meiotic recombination by DNA double-strand break formation is temporally coordinated with replication. Murakami and Keeney show that this coordination requires recruitment of the Dbf4-dependent kinase to the replication fork by the conserved TIM-TIPIN complex. The same mechanism may regulate other important replication-associated processes.

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Année de publication : 2013

Jesús A Carballo, Silvia Panizza, Maria Elisabetta Serrentino, Anthony L Johnson, Marco Geymonat, Valérie Borde, Franz Klein, Rita S Cha (2013 Jul 5)

Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery.

PLoS genetics : e1003545 : DOI : 10.1371/journal.pgen.1003545 En savoir plus
Résumé

An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or « DSB homeostasis », might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.

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Maria-Elisabetta Serrentino, Emmanuel Chaplais, Vérane Sommermeyer, Valérie Borde (2013 Apr 9)

Differential association of the conserved SUMO ligase Zip3 with meiotic double-strand break sites reveals regional variations in the outcome of meiotic recombination.

PLoS genetics : e1003416 : DOI : 10.1371/journal.pgen.1003416 En savoir plus
Résumé

During the first meiotic prophase, programmed DNA double-strand breaks (DSBs) are distributed non randomly at hotspots along chromosomes, to initiate recombination. In all organisms, more DSBs are formed than crossovers (CO), the repair product that creates a physical link between homologs and allows their correct segregation. It is not known whether all DSB hotspots are also CO hotspots or if the CO/DSB ratio varies with the chromosomal location. Here, we investigated the variations in the CO/DSB ratio by mapping genome-wide the binding sites of the Zip3 protein during budding yeast meiosis. We show that Zip3 associates with DSB sites that are engaged in repair by CO, and Zip3 enrichment at DSBs reflects the DSB tendency to be repaired by CO. Moreover, the relative amount of Zip3 per DSB varies with the chromosomal location, and specific chromosomal features are associated with high or low Zip3 per DSB. This work shows that DSB hotspots are not necessarily CO hotspots and suggests that different categories of DSB sites may fulfill different functions.

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Valérie Borde, Bernard de Massy (2013 Apr 2)

Programmed induction of DNA double strand breaks during meiosis: setting up communication between DNA and the chromosome structure.

Current opinion in genetics & development : 147-55 : DOI : 10.1016/j.gde.2012.12.002 En savoir plus
Résumé

During the first meiotic prophase, hundreds of DNA double strand breaks (DSBs) are deliberately self-inflicted along chromosomes in order to promote homologous recombination between homologs. These DSBs, catalyzed by the evolutionary conserved Spo11 protein, are highly regulated. Recent studies in yeast and mammals have identified key components involved in meiotic DSB formation. In mammals, the DNA binding specificity of PRDM9 determines where DSB occur, whereas in yeast, Spo11 acts in regions which one important feature is chromatin accessibility. However, DSB formation requires additional proteins located on chromosome axes, and the Saccharomyces cerevisiae protein, Spp1 has been recently identified to make the link between axes and DSB sites. These recent findings open exciting routes to understanding how the requirement to regulate DSBs along and between homologs is achieved.

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Vérane Sommermeyer, Claire Béneut, Emmanuel Chaplais, Maria Elisabetta Serrentino, Valérie Borde (2013 Jan 10)

Spp1, a member of the Set1 Complex, promotes meiotic DSB formation in promoters by tethering histone H3K4 methylation sites to chromosome axes.

Molecular cell : 43-54 : DOI : 10.1016/j.molcel.2012.11.008 En savoir plus
Résumé

Meiotic chromosomes are organized into arrays of loops that are anchored to the chromosome axis structure. Programmed DNA double-strand breaks (DSBs) that initiate meiotic recombination, catalyzed by Spo11 and accessory DSB proteins, form in loop sequences in promoters, whereas the DSB proteins are located on chromosome axes. Mechanisms bridging these two chromosomal regions for DSB formation have remained elusive. Here we show that Spp1, a conserved member of the histone H3K4 methyltransferase Set1 complex, is required for normal levels of DSB formation and is associated with chromosome axes during meiosis, where it physically interacts with the Mer2 DSB protein. The PHD finger module of Spp1, which reads H3K4 methylation close to promoters, promotes DSB formation by tethering these regions to chromosome axes and activating cleavage by the DSB proteins. This paper provides the molecular mechanism linking DSB sequences to chromosome axes and explains why H3K4 methylation is important for meiotic recombination.

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Année de publication : 2012

Maria-Elisabetta Serrentino, Valérie Borde (2012 Apr 11)

The spatial regulation of meiotic recombination hotspots: are all DSB hotspots crossover hotspots?

Experimental cell research : 1347-52 : DOI : 10.1016/j.yexcr.2012.03.025 En savoir plus
Résumé

A key step for the success of meiosis is programmed homologous recombination, during which crossovers, or exchange of chromosome arms, take place. Crossovers increase genetic diversity but their main function is to ensure accurate chromosome segregation. Defects in crossover number and position produce aneuploidies that represent the main cause of miscarriages and chromosomal abnormalities such as Down’s syndrome. Recombination is initiated by the formation of programmed double strand breaks (DSBs), which occur preferentially at places called DSB hotspots. Among all DSBs generated, only a small fraction is repaired by crossover, the other being repaired by other homologous recombination pathways. Crossover maps have been generated in a number of organisms, defining crossover hotspots. With the availability of genome-wide maps of DSBs as well as the ability to measure genetically the repair outcome at several hotspots, it is becoming more and more clear that not all DSB hotspots behave the same for crossover formation, suggesting that chromosomal features distinguish different types of hotspots.

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Elsa Brachet, Vérane Sommermeyer, Valérie Borde (2012 Feb 23)

Interplay between modifications of chromatin and meiotic recombination hotspots.

Biology of the cell / under the auspices of the European Cell Biology Organization : 51-69 : DOI : 10.1111/boc.201100113 En savoir plus
Résumé

Meiotic recombination lies at the heart of sexual reproduction. It is essential for producing viable gametes with a normal haploid genomic content and its dysfunctions can be at the source of aneuploidies, such as the Down syndrome, or many genetic disorders. Meiotic recombination also generates genetic diversity that is transmitted to progeny by shuffling maternal and paternal alleles along chromosomes. Recombination takes place at non-random chromosomal sites called ‘hotspots’. Recent evidence has shown that their location is influenced by properties of chromatin. In addition, many studies in somatic cells have highlighted the need for changes in chromatin dynamics to allow the process of recombination. In this review, we discuss how changes in the chromatin landscape may influence the recombination map, and reciprocally, how recombination events may lead to epigenetic modifications at sites of recombination, which could be transmitted to progeny.

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Année de publication : 2011

Emmanuelle Martini, Valérie Borde, Matthieu Legendre, Stéphane Audic, Béatrice Regnault, Guillaume Soubigou, Bernard Dujon, Bertrand Llorente (2011 Apr 28)

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

PLoS genetics : e1002305 : DOI : 10.1371/journal.pgen.1002305 En savoir plus
Résumé

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

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Année de publication : 2009

Hajime Murakami, Valérie Borde, Alain Nicolas, Scott Keeney (2009 Oct 6)

Gel electrophoresis assays for analyzing DNA double-strand breaks in Saccharomyces cerevisiae at various spatial resolutions.

Methods in molecular biology (Clifton, N.J.) : 117-42 : DOI : 10.1007/978-1-59745-527-5_9 En savoir plus
Résumé

Meiotic recombination is triggered by programmed DNA double-strand breaks (DSBs), which are catalyzed by Spo11 protein in a type II topoisomerase-like manner. Meiotic DSBs can be detected directly using physical assays (gel electrophoresis, Southern blotting, and indirect end-labeling) applied to samples of genomic DNA from sporulating cultures of budding and fission yeast. Such assays are extremely useful for quantifying and characterizing many aspects of the initiation of meiotic recombination, including the timing of DSB formation relative to other events, the distribution of DSBs across the genome, and the influence on DSB formation of mutations in recombination factors and other gene products. By varying the type of gel electrophoresis and other parameters, the spatial resolution of DSB analysis can range from single nucleotides up to whole yeast chromosomes.

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Valérie Borde, Nicolas Robine, Waka Lin, Sandrine Bonfils, Vincent Géli, Alain Nicolas (2009 Aug 6)

Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites.

The EMBO journal : 99-111 : DOI : 10.1038/emboj.2008.257 En savoir plus
Résumé

The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae, we examined the genome-wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double-strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild-type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB-prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.

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Valérie Borde, Jennifer Cobb (2009 Jan 20)

Double functions for the Mre11 complex during DNA double-strand break repair and replication.

The international journal of biochemistry & cell biology : 1249-53 : DOI : 10.1016/j.biocel.2008.12.013 En savoir plus
Résumé

Defining the factors that lead to genomic instability is one of the most important fields in cancer biology. DNA damage can arise from exogenous sources or as a result of normal cellular metabolism. Regardless of the cause, when damaged DNA is not properly repaired the genome acquires mutation(s). Under normal circumstances, to prevent such chromosome instability the cell activates the checkpoint response, which inhibits cell cycle progression until DNA repair is complete. The Mre11 complex is formed by three components: Mre11, Rad50, and Nbs1/Xrs2 and is involved in the signaling pathways that lead to both checkpoint activation and DNA repair. In response to DNA damage two functions of the complex will be discussed, one involves its role in initiating kinase activation and the second involves its ability to tether and link DNA strands. This review will highlight the functions of the Mre11 complex during the process of DNA double strand break recognition and repair, and during the process of replication. Understanding how the Mre11 complex is working at the molecular level is important for understanding why disruptions in components of the complex lead to genomic instability and cancer predisposition syndromes in humans.

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Année de publication : 2008

Ulrich Schlecht, Ionas Erb, Philippe Demougin, Nicolas Robine, Valérie Borde, Erik van Nimwegen, Alain Nicolas, Michael Primig (2008 Feb 29)

Genome-wide expression profiling, in vivo DNA binding analysis, and probabilistic motif prediction reveal novel Abf1 target genes during fermentation, respiration, and sporulation in yeast.

Molecular biology of the cell : 2193-207 : DOI : 10.1091/mbc.E07-12-1242 En savoir plus
Résumé

The autonomously replicating sequence binding factor 1 (Abf1) was initially identified as an essential DNA replication factor and later shown to be a component of the regulatory network controlling mitotic and meiotic cell cycle progression in budding yeast. The protein is thought to exert its functions via specific interaction with its target site as part of distinct protein complexes, but its roles during mitotic growth and meiotic development are only partially understood. Here, we report a comprehensive approach aiming at the identification of direct Abf1-target genes expressed during fermentation, respiration, and sporulation. Computational prediction of the protein’s target sites was integrated with a genome-wide DNA binding assay in growing and sporulating cells. The resulting data were combined with the output of expression profiling studies using wild-type versus temperature-sensitive alleles. This work identified 434 protein-coding loci as being transcriptionally dependent on Abf1. More than 60% of their putative promoter regions contained a computationally predicted Abf1 binding site and/or were bound by Abf1 in vivo, identifying them as direct targets. The present study revealed numerous loci previously unknown to be under Abf1 control, and it yielded evidence for the protein’s variable DNA binding pattern during mitotic growth and meiotic development.

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Année de publication : 2007

Rebecca Johnson, Valérie Borde, Matthew J Neale, Anna Bishop-Bailey, Matthew North, Sheila Harris, Alain Nicolas, Alastair S H Goldman (2007 Dec 18)

Excess single-stranded DNA inhibits meiotic double-strand break repair.

PLoS genetics : e223 En savoir plus
Résumé

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

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Valérie Borde (2007 Aug 4)

The multiple roles of the Mre11 complex for meiotic recombination.

Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology : 551-63 En savoir plus
Résumé

During the first meiotic prophase, numerous DNA double-strand breaks (DSB) are formed in the genome in order to initiate recombination between homologous chromosomes. The conserved Mre11 complex, formed of Mre11, Rad50 and Nbs1 (Xrs2 in Saccharomyces cerevisiae) proteins, plays a crucial role in mitotic cells for sensing and repairing DSB. In meiosis the Mre11 complex is also required for meiotic recombination. Depending on the organisms, the Mre11 complex is required for the formation of the DSB catalysed by the transesterase Spo11 protein. It then plays a unique function in removing covalently attached Spo11 from the 5′ extremity of the breaks through its nuclease activity, to allow further break resection. Finally, the Mre11 complex also plays a role during meiosis in bridging DNA molecules together and in sensing Spo11 DSB and activating the DNA damage checkpoint. In this article the different biochemical functions of the Mre11 complex required during meiosis are reviewed, as well as the consequences of Mre11 complex inactivation for meiosis in several organisms. Finally, I describe the meiotic phenotypes of several animal models that have been developed to model hypomorphic mutations of the Mre11 complex, involved in humans in some genetic instability disorders.

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