Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation

Jie Liu, Henning Paul-Julius Stahlberg
Springer Nature, 2011
Article

Résumé

Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyses the signature reactions of recombination, homology search and DNA strand invasion(1,2). Eukaryotes also possess Rad51 paralogues, whose exact role in recombination remains to be defined(3). Here we show that the Saccharomyces cerevisiae Rad51 paralogues, the Rad55-Rad57 heterodimer, counteract the antirecombination activity of the Srs2 helicase. The Rad55-Rad57 heterodimer associates with the Rad51-single-stranded DNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51-Rad55-Rad57 co-filament resists disruption by the Srs2 antirecombinase by blocking Srs2 translocation, involving a direct protein interaction between Rad55-Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogues in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 antirecombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55-Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogues, which are known to be involved in cancer predisposition and human disease.

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https://infoscience.epfl.ch/record/274458?ln=fr

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Jie Liu, Henning Paul-Julius Stahlberg
Springer Nature, 2011
Article

Résumé

Official source

https://infoscience.epfl.ch/record/274458?ln=fr

À propos de ce résultat

Concepts associés (13)

Concepts associés

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Publications associées (4)

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RADX: A novel single-stranded DNA-binding protein regulating telomere recombination

Anna-Sophia Briod

Telomeres are specialized nucleoprotein structures present at the ends of linear chromosome. The telomeric DNA part is comprised of 5-15 kilo base pairs of double stranded TTAGGG repeats and it contains at the 3' end a single-stranded G-rich overhang of 50-400 nucleotides. Telomeric DNA associates with a large number of proteins, which inhibit DNA damage checkpoint activation, DNA repair activities and nucleolytic degradation. To better understand the complex roles which telomeres play in genome stability, premature aging and cancer development we explored the telomeric proteomic environment with BioID. We employed a CRISPR/Cas9 based knock-in approach to integrate the promiscuous biotin ligase BirA at the genomic loci of the telomeric components TRF1, TRF2 and POT1. Thus, we expressed the fusion proteins from native promoters aiming at retaining native expression levels. Upon incubation with Biotin, the promiscuous biotinylase can label proximal proteins, which can be selectively purified and identified via mass spectrometry. Our BioID results indicated that TRF1, TRF2 and POT1 share a large number of common protein partners. In addition to already known telomere components, we identified a significant number of novel telomeric proteins. Among those newly identified proteins, we found RADX, an RPA-like single-stranded DNA-binding-protein, which counteracts RAD51 at stalled replication forks. Even though RADX had so far only been characterized for its role in DNA replication we observed colocalization of ectopically expressed 3xFlag-RADX with telomeres throughout the cell cycle. The colocalization decreased when the RADXs single-stranded (ss)DNA-binding domain was mutated. We therefore hypothesize that RADX binds the single-stranded G-rich telomeric strand either as displaced strand in the t-loop or as single-stranded telomeric overhang. Chromatin-immunoprecipitation experiments suggest that RADX plays a distinct role in telomere replication, since upon treatment with the replication stress inducing reagent hydroxyurea we observed increased binding of RADX specifically to telomeres. Notably, RADX depletion on its own did not alter telomere integrity and telomere length but upon co-depletion with POT1, telomere fragility, sister-chromatid associations and telomere length increased strikingly. POT1 and POT1/RADX depletions lead to increased associations of the recombinase RAD51 with telomeres and consequently silencing of RAD51 rescued telomere integrity and elongation implying the regulatory role of RADX for RAD51. In summary my thesis describes the proteomic microenvironment of different telomeric proteins, identifies the novel telomeric protein RADX and characterizes its role at telomeres. My findings provide important insights into how homologous recombination and RAD51 loading is suppressed at telomeres.

EPFL2019

Chargement

Identification and characterization of proteins that protect human telomeres from fragility

Anna Christina Näger

Difficulties to replicate telomeres - the ends of our chromosomes - can cause telomere shortening andgenome instability. These difficulties are due to the repetitive DNA sequence and distinct structures at telomeresthat challenge the semi-conservative DNA replication machinery. Among the proteins that help toovercome the obstacles to telomere replication are factors known to be mutated in genetic diseases (forinstance WRN, BLM, RTEL1, CTC1), but we suspect that there are still factors and pathways unknown.DNA replication defects at telomeres manifest as smeary or multiple telomeric fluorescence in situ hybridization(FISH) signals on metaphase chromosomes - commonly called telomere fragility. To identify proteinsthat protect human telomeres from fragility, we depleted 71 proteins (including 8 controls) from HeLa cellsusing siRNA pools and analysed their metaphase chromosomes for fragile telomeres. The majority of theseproteins had been identified by QTIP-iPOND - a proteomic analysis of chromatin near telomere replicationforks. Among the 71 proteins that were depleted in the siRNA screen, we identified 29 novel proteins thatprevent telomere fragility and are therefore crucial to maintain genome stability. The telomere fragilityscreen validated QTIP-iPOND as a technique to identify telomere replication proteins. The hits identified inour telomere fragility screen may for example have functions in regulating telomeric chromatin compositionand compaction, regulating transcription of the telomeric long non-coding RNA TERRA and its associationwith telomeric chromatin, dealing with replication stress and stalled replication forks, modulating DNAdamage signalling and repair, modulating sister chromatid cohesion, and preventing oxidative stress.One of the 29 novel proteins identified in the telomere fragility screen is chromobox 8 (CBX8) whose functionat telomeres was investigated in further detail in this study. CBX8 is a component of the canonical polycombrepressive complex 1 (PRC1), an epigenetic regulator of gene expression. However, CBX8 also hasfunctions that are independent of the PRC1 complex. We show that human CBX8 prevents telomere fragilitycaused by RNA-DNA hybrids which could stall replication forks and induce homologous recombination.CBX8 depletion or knockout did however not affect telomeric RNA-DNA hybrid levels. We hypothesize thatCBX8 may be involved in repair activities at stalled replication forks that arise when the replisome encounterspersistent RNA-DNA hybrids. Moreover, we found that CBX8 localizes to telomeric repeat-binding factor2 (TRF2)-deficient telomeres in a manner that is dependent on RNA-DNA hybrids and ATM signalling.We demonstrate that H3K27me3 (trimethylated histone 3 lysine 27) marks, which have previously beensuggested to help recruitment of CBX proteins to chromatin via the CBX chromodomain, are dispensable forCBX8 recruitment to TRF2-deficient telomeres. Furthermore, we found that CBX8 promotes non-homologousend joining (NHEJ)-mediated telomere fusions of dysfunctional telomeres. We therefore hypothesizethat CBX8 is involved in repair activities that influence the DNA repair pathway choice at TRF2-deficient telomeres.

EPFL2022

Chargement

Genome-wide manipulations and measurements have made huge progress over the last decades. In Saccharomyces cerevisiae, a well-studied eukaryotic model organism, homologous recombination allows for systematic deletion or alteration of a majority of its genes. Important products of these manipulation techniques are two libraries of modified strains: A deletion library consisting of all viable knockout mutants, and a GFP library in which 4159 proteins are successfully tagged with GFP. In addition, the development of a method that allows for the systematic construction of double mutants led to a virtually infinite number of potential strains of interest. These advancements in combinatorial biology need to be matched by methods of data measurement and analysis. In order to simultaneously observe the spatio-temporal dynamics of thousands of strains from the GFP library, Dénervaud et al. developed a microfluidic platform that allows for parallel imaging of 1152 strains in a single experiment. On this platform, strains can be grown and monitored in a controllable environment for several days, which results in the imaging of several millions of cells during one experiment. To objectively and quantitatively analyze this immense amount of information, we implemented an image analysis pipeline, which can extract experiment-wide information on single-cell protein abundance and subcellular localization. The construction of a supervised classifier to quantify localization information on a single cell level is a new approach and was invaluable to detect dynamic localization changes within the proteome. Using five different stress conditions, we gained insight into temporal changes of abundance and localization of multiple proteins. For example, we found that while localization changes can often be fast and transient, long-term response of a cell is usually enabled by changes in abundance. This shows a well-orchestrated response of a cell to external stimuli. To extend knowledge about cellular mechanisms, we used our microfluidic platform for two separate screens, combining GFP-reporter with additional deletion mutants. The advantage of our platform in comparison to more common approaches lies in its simultaneous measurement of fluorescence and phenotypic information on cell size and growth. For each deletion, we can quantify not only its influence onto the respective GFP-reporter under changing conditions, but also its effect on cell growth and size. We showed that it is advantageous to combine this information, as it allows pointing out possible underlying mechanisms of gene network regulations. In a first screen we investigated the behavior of several gene networks upon UV irradiation damage. We were able to show that four gene deletions influenced the localization of ribonucleotide-diphosphate reductase (Rnr4p). A second screen was designed to find genes that influence the induction of the galactose network. This screen uses more than 500 deletions of genes mostly related to chromatin in combination with two different reporter strains. A main focus of this study was the inheritance of memory during galactose reinduction. We found several previously unknown genes that potentially influence either induction or reinduction and were picked as candidates for further inheritance studies. Our microfluidic platform allows for unprecedented studies of proteomes in flux. [...]

EPFL2015

Chargement

EPFL2015

Identification and characterization of proteins that protect human telomeres from fragility

Anna Christina Näger

EPFL2022

RADX: A novel single-stranded DNA-binding protein regulating telomere recombination

Anna-Sophia Briod

EPFL2019