Asunto(s)
ADN , Telómero , Telómero/metabolismo , Humanos , ADN/metabolismo , ADN/química , Adenosina Difosfato/metabolismo , Telomerasa/metabolismoRESUMEN
Analysis of replication fork structures in electron microscopy (EM) can provide important mechanistic insights in DNA replication studies. A major challenge in this type of analysis is the paucity of replication intermediates. At any given time only a small fraction of the restriction fragments of genomic DNA will contain a replication fork. To address this issue, we have developed an EdU-pull-down procedure to enrich for replicating DNA. Cells are exposed to a brief pulse of EdU, a cleavable biotin moiety is attached to EdU by copper-catalyzed azide-alkyne cycloaddition (CuAAC), in conditions that minimize the damage to DNA. Biotinylated DNA is purified with streptavidin beads, in conditions that facilitate association of long DNA filaments. Finally, the DNA is eluted by cleaving the biotin moiety. This approach can enrich over 150-times for replicating DNA and about 50-times in replication fork structures, as verified by EM. This procedure could benefit analysis of replication intermediates in EM as well as other techniques for the study of replicating DNA.
Asunto(s)
Biotina , ADN , Biotina/química , ADN/genética , Replicación del ADNRESUMEN
Homologous recombination factors play a crucial role in protecting nascent DNA during DNA replication, but the role of chromatin in this process is largely unknown. Here, we used the bacterial Tus/Ter barrier known to induce a site-specific replication fork stalling in S. cerevisiae. We report that the Set1C subunit Spp1 is recruited behind the stalled replication fork independently of its interaction with Set1. Spp1 chromatin recruitment depends on the interaction of its PHD domain with H3K4me3 parental histones deposited behind the stalled fork. Its recruitment prevents the accumulation of ssDNA at the stalled fork by restricting the access of Exo1. We further show that deleting SPP1 increases the mutation rate upstream of the barrier favoring the accumulation of microdeletions. Finally, we report that Spp1 protects nascent DNA at the Tus/Ter stalled replication fork. We propose that Spp1 limits the remodeling of the fork, which ultimately limits nascent DNA availability to nucleases.
Asunto(s)
ADN , Saccharomyces cerevisiae , Núcleo Celular , Cromatina , Replicación del ADN , EndonucleasasRESUMEN
The telomerase reverse transcriptase elongates telomeres to prevent replicative senescence. This process requires exposure of the 3'-end, which is thought to occur when two sister telomeres are generated at replication completion. Using two-dimensional agarose gel electrophoresis (2D-gels) and electron microscopy, we found that telomeric repeats are hotspots for replication fork reversal. Fork reversal generates 3' telomeric ends before replication completion. To verify whether these ends are elongated by telomerase, we probed de novo telomeric synthesis in situ and at replication intermediates by reconstituting mutant telomerase that adds a variant telomere sequence. We found variant telomeric repeats overlapping with telomeric reversed forks in 2D-gels, but not with normal forks, nontelomeric reversed forks, or telomeric reversed forks with a C-rich 3'-end. Our results define reversed telomeric forks as a substrate of telomerase during replication.
Asunto(s)
Telomerasa , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismo , Replicación del ADNRESUMEN
Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells with structural chromosomal abnormalities that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures are upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.
Asunto(s)
Aberraciones Cromosómicas , Neoplasias , Humanos , Aneuploidia , Inestabilidad Genómica , Inestabilidad Cromosómica , Neoplasias/genética , Cariotipo , Segregación CromosómicaRESUMEN
Centromeres are key elements for chromosome segregation. Canonical centromeres are built over long-stretches of tandem repetitive arrays. Despite being quite abundant compared to other loci, centromere sequences overall still represent only 2 to 5% of the human genome, therefore studying their genetic and epigenetic features is a major challenge. Furthermore, sequencing of centromeric regions requires high coverage to fully analyze length and sequence variations, and this can be extremely costly. To bypass these issues, we have developed a technique, named CenRICH, to enrich for centromeric DNA from human cells based on selective restriction digestion and size fractionation. Combining restriction enzymes cutting at high frequency throughout the genome, except within most human centromeres, with size-selection of fragments >20 kb, resulted in over 25-fold enrichment in centromeric DNA. High-throughput sequencing revealed that up to 60% of the DNA in the enriched samples is made of centromeric repeats. We show that this method can be used in combination with long-read sequencing to investigate the DNA methylation status of certain centromeres and, with a specific enzyme combination, also of their surrounding regions (mainly HSATII). Finally, we show that CenRICH facilitates single-molecule analysis of replicating centromeric fibers by DNA combing. This approach has great potential for making sequencing of centromeric DNA more affordable and efficient and for single DNA molecule studies.
Asunto(s)
Centrómero , ADN , Centrómero/genética , Segregación Cromosómica , ADN/genética , HumanosRESUMEN
Here we provide a detailed protocol for the enrichment of telomeric repeats from mouse and human cells. The procedure consists of two successive rounds of digestion with frequently cutting restriction enzymes followed by size fractionation. Around 2 mg of genomic DNA is required, and the procedure lasts 5-6 d and yields preparations enriched >800-fold in telomeres. The purified material is suitable for single-molecule analysis of telomere structure, visualizing telomere replication and recombination intermediates by electron microscopy or performing molecular combing at telomeric repeats. No special skills are required for the enrichment procedure, while some assistance is needed in harvesting a large number of plates in a timely fashion at the beginning of the procedure. A smaller-scale version of the protocol that involves one round of digestion and purification requires 200 µg of DNA and enriches telomeres ~50-fold in 4 d is also provided. The latter can be combined with specific labeling for single-molecule analysis of replicating DNA or for long-read sequencing analysis of telomeric repeats. The procedure described here can be adapted to the enrichment of other repetitive elements, based on the use of restriction enzymes that do not cut into the repeat of interest.
Asunto(s)
ADN , Telómero , Animales , ADN/genética , Proteínas de Unión al ADN/metabolismo , Mamíferos/genética , Ratones , Secuencias Repetitivas de Ácidos Nucleicos , Telómero/genéticaRESUMEN
Extrachromosomal telomeric circles are commonly invoked as important players in telomere maintenance, but their origin has remained elusive. Using electron microscopy analysis on purified telomeres we show that, apart from known structures, telomeric repeats accumulate internal loops (i-loops) that occur in the proximity of nicks and single-stranded DNA gaps. I-loops are induced by single-stranded damage at normal telomeres and represent the majority of telomeric structures detected in ALT (Alternative Lengthening of Telomeres) tumor cells. Our data indicate that i-loops form as a consequence of the exposure of single-stranded DNA at telomeric repeats. Finally, we show that these damage-induced i-loops can be excised to generate extrachromosomal telomeric circles resulting in loss of telomeric repeats. Our results identify damage-induced i-loops as a new intermediate in telomere metabolism and reveal a simple mechanism that links telomere damage to the accumulation of extrachromosomal telomeric circles and to telomere erosion.
Asunto(s)
Telómero/química , Telómero/metabolismo , Animales , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Humanos , Ratones , Telómero/genética , Homeostasis del TelómeroRESUMEN
Telomeric repeats, coated by the shelterin complex, prevent inappropriate activation of the DNA damage response at the ends of linear chromosomes. Shelterin has evolved distinct solutions to protect telomeres from different aspects of the DNA damage response. These solutions include formation of t-loops, which can sequester the chromosome terminus from DNA-end sensors and inhibition of key steps in the DNA damage response. While blocking the DNA damage response at chromosome ends, telomeres make wide use of many of its players to deal with exogenous damage and replication stress. This review focuses on the interplay between the end-protection functions and the response to DNA damage occurring inside the telomeric repeats, as well as on the consequences that telomere damage has on telomere structure and function.
Asunto(s)
Reparación del ADN , Telómero/genética , Telómero/metabolismo , Animales , Daño del ADN , Humanos , Homeostasis del Telómero , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Shelterin protects chromosome ends from the DNA damage response. Although the mechanism of telomere protection has been studied extensively, the fate of double-strand breaks (DSBs) inside telomeres is not known. Here, we report that telomere-internal FokI-induced DSBs activate ATM kinase-dependent signaling in S-phase but are well tolerated and repaired efficiently. Homologous recombination contributes to repair, leading to increased telomere length heterogeneity typical of the alternative lengthening of telomeres (ALT) pathway. Furthermore, cells accumulate extra chromosomal telomeric signals (ECTS), a second hallmark of ALT. Telomere-internal DSBs are also repaired by a PARP1- and Ligase3-dependent reaction, suggesting alternative non-homologous end-joining (alt-NHEJ), which relies on microhomology at DSBs. However, as resected telomere-internal DSBs have perfect homology, their PARP1/Lig3-dependent end-joining may be more akin to single strand break repair. We conclude that shelterin does not repress ATM kinase signaling or DSB repair at telomere-internal sites, thereby allowing DNA repair to maintain telomere integrity.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP)/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Reparación del ADN por Recombinación , Telómero/metabolismo , Animales , Ratones , Modelos Biológicos , Fase S , Transducción de SeñalRESUMEN
Telomeres have evolved to protect the ends of linear chromosomes from the myriad of threats posed by the cellular DNA damage signaling and repair pathways. Mammalian telomeres have to block nonhomologous end joining (NHEJ), thus preventing chromosome fusions; they need to control homologous recombination (HR), which could change telomere lengths; they have to avoid activating the ATM (ataxia telangiectasia mutated) and ATR (ATM- and RAD3-related) kinase pathways, which could induce cell cycle arrest; and they have to protect chromosome ends from hyperresection. Recent studies of telomeres have provided insights into the mechanisms of NHEJ and HR, how these double-strand break (DSB) repair pathways can be thwarted, and how telomeres have co-opted DNA repair factors to help in the protection of chromosome ends. These aspects of telomere biology are reviewed here with particular emphasis on recombination, the main focus of this collection.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Evolución Molecular , Recombinación Homóloga/fisiología , Modelos Genéticos , Telómero/fisiología , Animales , Reparación del ADN por Unión de Extremidades/fisiología , Humanos , Complejo Shelterina , Proteínas de Unión a Telómeros/metabolismoRESUMEN
We have applied a super-resolution fluorescence imaging method, stochastic optical reconstruction microscopy (STORM), to visualize the structure of functional telomeres and telomeres rendered dysfunctional through removal of shelterin proteins. The STORM images showed that functional telomeres frequently exhibit a t-loop configuration. Conditional deletion of individual components of shelterin showed that TRF2 was required for the formation and/or maintenance of t-loops, whereas deletion of TRF1, Rap1, or the POT1 proteins (POT1a and POT1b) had no effect on the frequency of t-loop occurrence. Within the shelterin complex, TRF2 uniquely serves to protect telomeres from two pathways that are initiated on free DNA ends: classical nonhomologous end-joining (NHEJ) and ATM-dependent DNA damage signaling. The TRF2-dependent remodeling of telomeres into t-loop structures, which sequester the ends of chromosomes, can explain why NHEJ and the ATM signaling pathway are repressed when TRF2 is present.
Asunto(s)
Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Ratones , Microscopía Fluorescente , Complejo Shelterina , Proteínas de Unión a TelómerosRESUMEN
Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.
Asunto(s)
Replicación del ADN , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Quinasa de Punto de Control 2 , Roturas del ADN de Doble Cadena , Hidroxiurea/farmacología , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
In response to replication stress, the Mec1/ATR and SUMO pathways control stalled- and damaged-fork stability. We investigated the S phase response at forks encountering a broken template (termed the terminal fork). We show that double-strand break (DSB) formation can locally trigger dormant origin firing. Irreversible fork resolution at the break does not impede progression of the other fork in the same replicon (termed the sister fork). The Mre11-Tel1/ATM response acts at terminal forks, preventing accumulation of cruciform DNA intermediates that tether sister chromatids and can undergo nucleolytic processing. We conclude that sister forks can be uncoupled during replication and that, after DSB-induced fork termination, replication is rescued by dormant origin firing or adjacent replicons. We have uncovered a Tel1/ATM- and Mre11-dependent response controlling terminal fork integrity. Our findings have implications for those genome instability syndromes that accumulate DNA breaks during S phase and for forks encountering eroding telomeres.
Asunto(s)
Replicación del ADN , Replicón , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Origen de Réplica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600-base-pair (bp) region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M-G1 transition. top1 top2 double mutants exhibit fork block and processing and phosphorylation of Rad53 and gamma H2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation, and chromosome breakage during segregation.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN de Hongos/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Secuencia de Consenso , Daño del ADN , Replicación del ADN , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo II/genética , ADN de Hongos/química , ADN de Hongos/genética , Genes Fúngicos , Modelos Biológicos , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.