RESUMEN
Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that â¼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells. We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites to facilitate repair.
Asunto(s)
Arabidopsis/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , ARN de Planta/metabolismo , ARN no Traducido/metabolismo , ARN Helicasas DEAD-box/metabolismo , Humanos , Ribonucleasa III/metabolismoRESUMEN
Human telomeres are protected from DNA damage by a nucleoprotein complex that includes the repeat-binding factor TRF2. Here, we report that TRF2 regulates the 5' exonuclease activity of its binding partner, Apollo, a member of the metallo-beta-lactamase family that is required for telomere integrity during S phase. TRF2 and Apollo also suppress damage to engineered interstitial telomere repeat tracts that were inserted far away from chromosome ends. Genetic data indicate that DNA topoisomerase 2alpha acts in the same pathway of telomere protection as TRF2 and Apollo. Moreover, TRF2, which binds preferentially to positively supercoiled DNA substrates, together with Apollo, negatively regulates the amount of TOP1, TOP2alpha, and TOP2beta at telomeres. Our data are consistent with a model in which TRF2 and Apollo relieve topological stress during telomere replication. Our work also suggests that cellular senescence may be caused by topological problems that occur during the replication of the inner portion of telomeres.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Replicación del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Senescencia Celular , Daño del ADN , Exodesoxirribonucleasas , Humanos , Estructura Terciaria de ProteínaRESUMEN
In animals, distant H3K27me3-marked Polycomb targets can establish physical interactions forming repressive chromatin hubs. In plants, growing evidence suggests that H3K27me3 acts directly or indirectly to regulate chromatin interactions, although how this histone modification modulates 3D chromatin architecture remains elusive. To decipher the impact of the dynamic deposition of H3K27me3 on the Arabidopsis thaliana nuclear interactome, we combined genetics, transcriptomics, and several 3D epigenomic approaches. By analyzing mutants defective for histone H3K27 methylation or demethylation, we uncovered the crucial role of this chromatin mark in short- and previously unnoticed long-range chromatin loop formation. We found that a reduction in H3K27me3 levels led to a decrease in the interactions within Polycomb-associated repressive domains. Regions with lower H3K27me3 levels in the H3K27 methyltransferase clf mutant established new interactions with regions marked with H3K9ac, a histone modification associated with active transcription, indicating that a reduction in H3K27me3 levels induces a global reconfiguration of chromatin architecture. Altogether, our results reveal that the 3D genome organization is tightly linked to reversible histone modifications that govern chromatin interactions. Consequently, nuclear organization dynamics shapes the transcriptional reprogramming during plant development and places H3K27me3 as a key feature in the coregulation of distant genes.
RESUMEN
Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , ADN Helicasas/fisiología , Recombinasa Rad51/fisiología , Acortamiento del Telómero , Arabidopsis/enzimología , Proteínas de Arabidopsis/análisis , Replicación del ADN , Inestabilidad Genómica , Recombinación Homóloga , Mutación , Recombinasa Rad51/análisis , Secuencias Repetitivas de Ácidos Nucleicos , Ribonucleasas/genética , Procesos Estocásticos , Telomerasa/genética , Telómero/químicaRESUMEN
Despite intensive searches, few proteins involved in telomere homeostasis have been identified in plants. Here, we used pull-down assays to identify potential telomeric interactors in the model plant species Arabidopsis (Arabidopsis thaliana). We identified the candidate protein GH1-HMGA1 (also known as HON4), an uncharacterized linker histone protein of the High Mobility Group Protein A (HMGA) family in plants. HMGAs are architectural transcription factors and have been suggested to function in DNA damage repair, but their precise biological roles remain unclear. Here, we show that GH1-HMGA1 is required for efficient DNA damage repair and telomere integrity in Arabidopsis. GH1-HMGA1 mutants exhibit developmental and growth defects, accompanied by ploidy defects, increased telomere dysfunction-induced foci, mitotic anaphase bridges, and degraded telomeres. Furthermore, mutants have a higher sensitivity to genotoxic agents such as mitomycin C and γ-irradiation. Our work also suggests that GH1-HMGA1 is involved directly in the repair process by allowing the completion of homologous recombination.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Daño del ADN , Reparación del ADN , Proteínas HMGA/metabolismo , Histonas/metabolismo , Telómero/metabolismo , Arabidopsis/crecimiento & desarrollo , Cromatina/metabolismo , ADN Bacteriano/genética , Fluorescencia , Recombinación Homóloga/genética , Mutación/genética , Unión Proteica , Telomerasa/metabolismo , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Structure-specific endonucleases act to repair potentially toxic structures produced by recombination and DNA replication, ensuring proper segregation of the genetic material to daughter cells during mitosis and meiosis. Arabidopsis thaliana has two putative homologs of the resolvase (structure-specific endonuclease): GEN1/Yen1. Knockout of resolvase genes GEN1 and SEND1, individually or together, has no detectable effect on growth, fertility, or sensitivity to DNA damage. However, combined absence of the endonucleases MUS81 and SEND1 results in severe developmental defects, spontaneous cell death, and genome instability. A similar effect is not seen in mus81 gen1 plants, which develop normally and are fertile. Absence of RAD51 does not rescue mus81 send1, pointing to roles of these proteins in DNA replication rather than DNA break repair. The enrichment of S-phase histone γ-H2AX foci and a striking loss of telomeric DNA in mus81 send1 further support this interpretation. SEND1 has at most a minor role in resolution of the Holliday junction but acts as an essential backup to MUS81 for resolution of toxic replication structures to ensure genome stability and to maintain telomere integrity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endonucleasas/metabolismo , Resolvasas de Unión Holliday/metabolismo , Telómero/metabolismo , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Ciclo Celular , Cromosomas de las Plantas/genética , Reparación del ADN , Replicación del ADN , ADN Bacteriano/genética , Inestabilidad Genómica , Meiosis , Mutagénesis Insercional/genética , Mutación/genética , Fenotipo , Recombinasa Rad51/metabolismoRESUMEN
The WEE1 kinase is an essential cell cycle checkpoint regulator in Arabidopsis thaliana plants experiencing replication defects. Whereas under non-stress conditions WEE1-deficient plants develop normally, they fail to adapt to replication inhibitory conditions, resulting in the accumulation of DNA damage and loss of cell division competence. We identified mutant alleles of the genes encoding subunits of the ribonuclease H2 (RNase H2) complex, known for its role in removing ribonucleotides from DNA-RNA duplexes, as suppressor mutants of WEE1 knockout plants. RNase H2 deficiency triggered an increase in homologous recombination (HR), correlated with the accumulation of γ-H2AX foci. However, as HR negatively impacts the growth of WEE1-deficient plants under replication stress, it cannot account for the rescue of the replication defects of the WEE1 knockout plants. Rather, the observed increase in ribonucleotide incorporation in DNA indicates that the substitution of deoxynucleotide with ribonucleotide abolishes the need for WEE1 under replication stress. Strikingly, increased ribonucleotide incorporation in DNA correlated with the occurrence of small base pair deletions, identifying the RNase H2 complex as an important suppressor of genome instability.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Puntos de Control del Ciclo Celular , Inestabilidad Genómica , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleasa H/deficiencia , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Emparejamiento Base , Secuencia de Bases , Dominio Catalítico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Genes de Plantas , Inestabilidad Genómica/efectos de los fármacos , Hidroxiurea/farmacología , Datos de Secuencia Molecular , Mutación/genética , Tasa de Mutación , Recombinación Genética/genética , Ribonucleasa H/química , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Ribonucleótidos/metabolismoRESUMEN
Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Factor 1 de Ensamblaje de la Cromatina/metabolismo , ADN Ribosómico/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Factor 1 de Ensamblaje de la Cromatina/genética , Reparación del ADN/genética , Reparación del ADN/fisiología , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiologíaRESUMEN
The telomeres of linear eukaryotic chromosomes are protected by caps consisting of evolutionarily conserved nucleoprotein complexes. Telomere dysfunction leads to recombination of chromosome ends and this can result in fusions which initiate chromosomal breakage-fusion-bridge cycles, causing genomic instability and potentially cell death or cancer. We hypothesize that in the absence of the recombination pathways implicated in these fusions, deprotected chromosome ends will instead be eroded by nucleases, also leading to the loss of genes and cell death. In this work, we set out to specifically test this hypothesis in the plant, Arabidopsis. Telomere protection in Arabidopsis implicates KU and CST and their absence leads to chromosome fusions, severe genomic instability and dramatic developmental defects. We have analysed the involvement of end-joining recombination pathways in telomere fusions and the consequences of this on genomic instability and growth. Strikingly, the absence of the multiple end-joining pathways eliminates chromosome fusion and restores normal growth and development to cst ku80 mutant plants. It is thus the chromosomal fusions, per se, which are the underlying cause of the severe developmental defects. This rescue is mediated by telomerase-dependent telomere extension, revealing a competition between telomerase and end-joining recombination proteins for access to deprotected telomeres.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Reparación del ADN por Unión de Extremidades , Recombinación Genética , Telomerasa/fisiología , Homeostasis del Telómero , Proteínas de Unión a Telómeros/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , ADN Helicasas/genética , Reparación del ADN , Mutación , Secuencias Repetitivas de Ácidos Nucleicos , Telomerasa/genética , Telómero/química , Acortamiento del TelómeroRESUMEN
Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Meiosis , Recombinasa Rad51/genética , Rec A Recombinasas/genética , Recombinación Genética/genética , Arabidopsis , Cromosomas/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismoRESUMEN
Homologous recombination is key to the maintenance of genome integrity and the creation of genetic diversity. At the mechanistic level, recombination involves the invasion of a homologous DNA template by broken DNA ends, repair of the break and exchange of genetic information between the two DNA molecules. Invasion of the template in eukaryotic cells is catalysed by the RAD51 and DMC1 recombinases, assisted by a number of accessory proteins, including the RAD51 paralogues. Eukaryotic genomes encode a variable number of RAD51 paralogues, ranging from two in yeast to five in animals and plants. The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilization of the RAD51-DNA nucleofilament. Somatic recombination assays and immunocytology confirm that the three 'non-meiotic' paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and that they are not required for the formation of radioinduced RAD51 foci. Given the presence of all five proteins in meiotic cells, the apparent absence of a meiotic role for RAD51B, RAD51D and XRCC2 is surprising, and perhaps simply the result of a more subtle meiotic phenotype in the mutants. Analysis of meiotic recombination confirms this, showing that the absence of XRCC2, and to a lesser extent RAD51B, but not RAD51D, increases rates of meiotic crossing over. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Unión al ADN/genética , Recombinación Homóloga/genética , Recombinasa Rad51/genética , Animales , Arabidopsis/efectos de los fármacos , Bleomicina/farmacología , Núcleo Celular/genética , ADN de Plantas/genética , ADN de Plantas/metabolismo , ADN de Plantas/farmacología , Mutación INDEL , Meiosis/genética , Mitosis/genética , Fenotipo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genéticaRESUMEN
The ends of linear eukaryotic chromosomes are hidden in nucleoprotein structures called telomeres, and loss of the telomere structure causes inappropriate repair, leading to severe karyotypic and genomic instability. Although it has been shown that DNA damaging agents activate a DNA damage response (DDR), little is known about the signaling of dysfunctional plant telomeres. We show that absence of telomerase in Arabidopsis thaliana elicits an ATAXIA-TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR)-dependent DDR at telomeres, principally through ATM. By contrast, telomere dysfunction induces an ATR-dependent response in telomeric Conserved telomere maintenance component1 (Ctc1)-Suppressor of cdc thirteen (Stn1)-Telomeric pathways in association with Stn1 (CST)-complex mutants. These results uncover a new role for the CST complex in repressing the ATR-dependent DDR pathway in plant cells and show that plant cells use two different DNA damage surveillance pathways to signal telomere dysfunction. The absence of either ATM or ATR in ctc1 and stn1 mutants significantly enhances developmental and genome instability while reducing stem cell death. These data thus give a clear illustration of the action of ATM/ATR-dependent programmed cell death in maintaining genomic integrity through elimination of genetically unstable cells.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinasas/genética , Telómero/genética , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Muerte Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de las Plantas/genética , Cromosomas de las Plantas/metabolismo , Daño del ADN , Activación Enzimática , Genoma de Planta , Inestabilidad Genómica , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Células Vegetales/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Telómero/metabolismo , Homeostasis del Telómero , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA.
Asunto(s)
Telómero/química , Proteína 1 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/química , Secuencia de Aminoácidos , ADN/química , ADN/metabolismo , Evolución Molecular , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN/metabolismo , Homología de Secuencia de Aminoácido , Telómero/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
Plant-specific transcriptional regulators called TELOMERE REPEAT BINDING proteins (TRBs) combine two DNA-binding domains, the GH1 domain, which binds to linker DNA and is shared with H1 histones, and the Myb/SANT domain, which specifically recognizes the telobox DNA-binding site motif. TRB1, TRB2, and TRB3 proteins recruit Polycomb group complex 2 (PRC2) to deposit H3K27me3 and JMJ14 to remove H3K4me3 at gene promoters containing telobox motifs to repress transcription. Here, we demonstrate that TRB4 and TRB5, two related paralogs belonging to a separate TRB clade conserved in spermatophytes, regulate the transcription of several hundred genes involved in developmental responses to environmental cues. TRB4 binds to several thousand sites in the genome, mainly at transcription start sites and promoter regions of transcriptionally active and H3K4me3-marked genes, but, unlike TRB1, it is not enriched at H3K27me3-marked gene bodies. However, TRB4 can physically interact with the catalytic components of PRC2, SWINGER, and CURLY LEAF (CLF). Unexpectedly, we show that TRB4 and TRB5 are required for distinctive phenotypic traits observed in clf mutant plants and thus function as transcriptional activators of several hundred CLF-controlled genes, including key flowering genes. We further demonstrate that TRB4 shares multiple target genes with TRB1 and physically and genetically interacts with members of both TRB clades. Collectively, these results reveal that TRB proteins engage in both positive and negative interactions with other members of the family to regulate plant development through both PRC2-dependent and -independent mechanisms.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Desarrollo de la Planta/genética , Proteínas de HomeodominioRESUMEN
The ability of the telomeric DNA-binding protein, TRF2, to stimulate t-loop formation while preventing t-loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t-loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t-loops and at regressed replication forks.
Asunto(s)
ADN Cruciforme/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Bacterias/enzimología , Emparejamiento Base , Secuencia de Bases , Bioensayo , Histidina/metabolismo , Resolvasas de Unión Holliday/metabolismo , Humanos , Datos de Secuencia Molecular , Permanganato de Potasio/farmacología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Recombinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Proteína 2 de Unión a Repeticiones Teloméricas/químicaRESUMEN
Signaling of chromosomal DNA breaks is of primary importance for initiation of repair and, thus, for global genomic stability. Although the Mre11-Rad50-Nbs1 (MRN) complex is the first sensor of double-strand breaks, its role in double-strand break (DSB) signaling is not fully understood. We report the absence of γ-ray-induced, ATM/ATR-dependent histone H2AX phosphorylation in Arabidopsis thaliana rad50 and mre11 mutants, confirming that the MRN complex is required for H2AX phosphorylation by the ATM and ATR kinases in response to irradiation-induced DSB in Arabidopsis. rad50 and mre11 mutants spontaneously activate a DNA damage response, as shown by the presence of γ-H2AX foci and activation of cell cycle arrest in nonirradiated plants. This response is ATR dependent as shown both by the absence of these spontaneous foci and by the wild-type mitotic indices of double rad50 atr and mre11 atr plants. EdU S-phase labeling and fluorescence in situ hybridization analysis using specific subtelomeric probes point to a replicative S-phase origin of this chromosome damage in the double mutants and not to telomere destabilization. Thus, the data presented here show the exclusive involvement of ATR in DNA damage signaling in MRN mutants and provide evidence for a role for ATR in the avoidance of S-phase DNA damage.
Asunto(s)
Arabidopsis/enzimología , Inestabilidad Cromosómica , Daño del ADN , Histonas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Rayos gamma , Proteína Homóloga de MRE11 , Mutagénesis Insercional , FosforilaciónRESUMEN
While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we show that, in Arabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2 cis recruitment. This study provides a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Animales , Histonas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cromatina , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/genética , Telómero/metabolismo , Mamíferos/metabolismoRESUMEN
Telomeres can fold into t-loops that may result from the invasion of the 3' overhang into duplex DNA. Their formation is facilitated in vitro by the telomeric protein TRF2, but very little is known regarding the mechanisms involved. Here we reveal that TRF2 generates positive supercoiling and condenses DNA. Using a variety of TRF2 mutants, we demonstrate a strong correlation between this topological activity and the ability to stimulate strand invasion. We also report that these properties require the combination of the TRF-homology (TRFH) domain of TRF2 with either its N- or C-terminal DNA-binding domains. We propose that TRF2 complexes, by constraining DNA around themselves in a right-handed conformation, can induce untwisting of the neighboring DNA, thereby favoring strand invasion. Implications of this topological model in t-loop formation and telomere homeostasis are discussed.
Asunto(s)
ADN/química , Telómero/química , Proteína 2 de Unión a Repeticiones Teloméricas/química , ADN Superhelicoidal/química , Humanos , Microscopía de Fuerza Atómica , Conformación de Ácido Nucleico , Proteína 1 de Unión a Repeticiones Teloméricas/químicaRESUMEN
Telomeres, the nucleoprotein complexes located at the ends of chromosomes, are involved in chromosome protection and genome stability. Telomeric repeat binding factor 1 (TRF1) and telomeric repeat binding factor 2 (TRF2) are the two telomeric proteins that bind to duplex telomeric DNA through interactions between their C-terminal domain and several guanines of the telomeric tract. Since the antitumour drug cisplatin binds preferentially to two adjacent guanines, we have investigated whether cisplatin adducts could affect the binding of TRF1 and TRF2 to telomeric DNA and the property of TRF2 to stimulate telomeric invasion, a process that is thought to participate in the formation of the t-loop. We show that the binding of TRF1 and TRF2 to telomeric sequences selectively modified by one GG chelate of cisplatin is markedly affected by cisplatin but that the effect is more drastic for TRF2 than for TRF1 (3-5-fold more sensitivity for TRF2 than for TRF1). We also report that platinum adducts cause a decrease in TRF2-dependent stimulation of telomeric invasion in vitro. Finally, in accordance with in vitro data, analysis of telomeric composition after cisplatin treatment reveals that 60% of TRF2 dissociate from telomeres.
Asunto(s)
Cisplatino/química , Cisplatino/farmacología , ADN/química , ADN/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Sitios de Unión/efectos de los fármacos , Línea Celular , ADN/síntesis química , Humanos , Unión Proteica/efectos de los fármacos , Complejo Shelterina , Telómero/metabolismoRESUMEN
A major issue in telomere research is to understand how the integrity of chromosome ends is preserved . The human telomeric protein TRF2 coordinates several pathways that prevent checkpoint activation and chromosome fusions. In this work, we identified hSNM1B, here named Apollo, as a novel TRF2-interacting factor. Interestingly, the N-terminal domain of Apollo is closely related to that of Artemis, a factor involved in V(D)J recombination and DNA repair. Both proteins belong to the beta-CASP metallo-beta-lactamase family of DNA caretaker proteins. Apollo appears preferentially localized at telomeres in a TRF2-dependent manner. Reduced levels of Apollo exacerbate the sensitivity of cells to TRF2 inhibition, resulting in severe growth defects and an increased number of telomere-induced DNA-damage foci and telomere fusions. Purified Apollo protein exhibits a 5'-to-3' DNA exonuclease activity. We conclude that Apollo is a novel component of the human telomeric complex and works together with TRF2 to protect chromosome termini from being recognized and processed as DNA damage. These findings unveil a previously undescribed telomere-protection mechanism involving a DNA 5'-to-3' exonuclease.