RESUMEN
Hi-C, a genome-wide chromosome conformation capture assay, is a powerful tool used to study three-dimensional genome organization by converting physical pairwise interactions into counts of pairwise interactions. To study the many temporally regulated facets of meiotic recombination in S. cerevisiae, the Hi-C assay must be robust such that fine- and wide-scale comparisons between genetic datasets can be made. Here we describe an updated protocol for Hi-C (Hi-C2B) that generates reproducible libraries of interaction data with low noise and for a relatively low cost.
Asunto(s)
Cromosomas Fúngicos , Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Meiosis/genética , Cromosomas Fúngicos/genética , Recombinación Genética , Genoma FúngicoRESUMEN
During meiosis, Spo11 generates DNA double-strand breaks to induce recombination, becoming covalently attached to the 5' ends on both sides of the break during this process. Such Spo11 "covalent complexes" are transient in wild-type cells, but accumulate in nuclease mutants unable to initiate repair. The CC-seq method presented here details how to map the location of these Spo11 complexes genome-wide with strand-specific nucleotide-resolution accuracy in synchronized Saccharomyces cerevisiae meiotic cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Meiosis/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Análisis de Secuencia de ADN/métodos , Reparación del ADNRESUMEN
DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an SLX4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements.
Asunto(s)
ADN-Topoisomerasas de Tipo II , Heterocromatina , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Heterocromatina/metabolismo , Animales , Inhibidores de Topoisomerasa II/farmacología , Secuencias Repetitivas de Ácidos Nucleicos/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Replicación del ADN , ADN Superhelicoidal/metabolismo , ADN Superhelicoidal/química , Humanos , Ratones , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN/metabolismo , ADN/química , InterfaseRESUMEN
During meiosis, genetic recombination is initiated by the formation of many DNA double-strand breaks (DSBs) catalysed by the evolutionarily conserved topoisomerase-like enzyme, Spo11, in preferred genomic sites known as hotspots. DSB formation activates the Tel1/ATM DNA damage responsive (DDR) kinase, locally inhibiting Spo11 activity in adjacent hotspots via a process known as DSB interference. Intriguingly, in S. cerevisiae, over short genomic distances (<15 kb), Spo11 activity displays characteristics of concerted activity or clustering, wherein the frequency of DSB formation in adjacent hotspots is greater than expected by chance. We have proposed that clustering is caused by a limited number of sub-chromosomal domains becoming primed for DSB formation. Here, we provide evidence that DSB clustering is abolished when meiotic prophase timing is extended via deletion of the NDT80 transcription factor. We propose that extension of meiotic prophase enables most cells, and therefore most chromosomal domains within them, to reach an equilibrium state of similar Spo11-DSB potential, reducing the impact that priming has on estimates of coincident DSB formation. Consistent with this view, when Tel1 is absent but Ndt80 is present and thus cells are able to rapidly exit meiotic prophase, genome-wide maps of Spo11-DSB formation are skewed towards pericentromeric regions and regions that load pro-DSB factors early-revealing regions of preferential priming-but this effect is abolished when NDT80 is deleted. Our work highlights how the stochastic nature of Spo11-DSB formation in individual cells within the limited temporal window of meiotic prophase can cause localised DSB clustering-a phenomenon that is exacerbated in tel1Δ cells due to the dual roles that Tel1 has in DSB interference and meiotic prophase checkpoint control.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Saccharomyces cerevisiae , ADN , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Meiosis/genética , Profase/genética , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Meiotic recombination shows broad variations across species and along chromosomes and is often suppressed at and around genomic regions determining sexual compatibility such as mating type loci in fungi. Here, we show that the absence of Spo11-DSBs and meiotic recombination on Lakl0C-left, the chromosome arm containing the sex locus of the Lachancea kluyveri budding yeast, results from the absence of recruitment of the two chromosome axis proteins Red1 and Hop1, essential for proper Spo11-DSBs formation. Furthermore, cytological observation of spread pachytene meiotic chromosomes reveals that Lakl0C-left does not undergo synapsis. However, we show that the behavior of Lakl0C-left is independent of its particularly early replication timing and is not accompanied by any peculiar chromosome structure as detectable by Hi-C in this yet poorly studied yeast. Finally, we observed an accumulation of heterozygous mutations on Lakl0C-left and a sexual dimorphism of the haploid meiotic offspring, supporting a direct effect of this absence of meiotic recombination on L. kluyveri genome evolution and fitness. Because suppression of meiotic recombination on sex chromosomes is widely observed across eukaryotes, the mechanism for recombination suppression described here may apply to other species, with the potential to impact sex chromosome evolution.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Cromosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Recombinación Homóloga/genética , Meiosis/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix-turn-helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a localized reduction of axis protein binding and meiotic DNA double-strand breaks (DSBs) in axis islands and leads to defects in chromosome synapsis. Synthetic effects with mutants of the Hop1 regulator Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of the CBR, suggesting that the mechanisms we uncover are broadly conserved.
Asunto(s)
Meiosis , Proteínas de Saccharomyces cerevisiae , Nucleosomas , Microscopía por Crioelectrón , Filogenia , Saccharomyces cerevisiae/genética , ADN , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
DNA double-strand break (DSB) repair by homologous recombination (HR) uses a DNA template with similar sequence to restore genetic identity. Allelic DNA repair templates can be found on the sister chromatid or homologous chromosome. During meiotic recombination, DSBs preferentially repair from the homologous chromosome, with a proportion of HR events generating crossovers. Nevertheless, regions of similar DNA sequence exist throughout the genome, providing potential DNA repair templates. When DSB repair occurs at these non-allelic loci (termed ectopic recombination), chromosomal duplications, deletions and rearrangements can arise. Here, we characterize in detail ectopic recombination arising between a dispersed pair of inverted repeats in wild-type Saccharomyces cerevisiae at both a local and a chromosomal scale-the latter identified via gross chromosomal acentric and dicentric chromosome rearrangements. Mutation of the DNA damage checkpoint clamp loader Rad24 and the RecQ helicase Sgs1 causes an increase in ectopic recombination. Unexpectedly, additional mutation of the RecA orthologues Rad51 and Dmc1 alters-but does not abolish-the type of ectopic recombinants generated, revealing a novel class of inverted chromosomal rearrangement driven by the single-strand annealing pathway. These data provide important insights into the role of key DNA repair proteins in regulating DNA repair pathway and template choice during meiosis.
Asunto(s)
Reparación del ADN , Meiosis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Proteínas de Ciclo Celular/metabolismo , Aberraciones Cromosómicas , ADN/metabolismo , Roturas del ADN de Doble Cadena , Recombinasas/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: The main function of telomerase is at the telomeres but under adverse conditions telomerase can bind to internal regions causing deleterious effects as observed in cancer cells. RESULTS: By mapping the global occupancy of the catalytic subunit of telomerase (Est2) in the budding yeast Saccharomyces cerevisiae, we reveal that it binds to multiple guanine-rich genomic loci, which we termed "non-telomeric binding sites" (NTBS). We characterize Est2 binding to NTBS. Contrary to telomeres, Est2 binds to NTBS in G1 and G2 phase independently of Est1 and Est3. The absence of Est1 and Est3 renders telomerase inactive at NTBS. However, upon global DNA damage, Est1 and Est3 join Est2 at NTBS and telomere addition can be observed indicating that Est2 occupancy marks NTBS regions as particular risks for genome stability. CONCLUSIONS: Our results provide a novel model of telomerase regulation in the cell cycle using internal regions as "parking spots" of Est2 but marking them as hotspots for telomere addition.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Telomerasa , Daño del ADN , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genéticaRESUMEN
Genetic recombination arises during meiosis through the repair of DNA double-strand breaks (DSBs) that are created by Spo11, a topoisomerase-like protein1,2. Spo11 DSBs form preferentially in nucleosome-depleted regions termed hotspots3,4, yet how Spo11 engages with its DNA substrate to catalyse DNA cleavage is poorly understood. Although most recombination events are initiated by a single Spo11 cut, here we show in Saccharomyces cerevisiae that hyperlocalized, concerted Spo11 DSBs separated by 33 to more than 100 base pairs also form, which we term 'double cuts'. Notably, the lengths of double cuts vary with a periodicity of 10.5 base pairs, which is conserved in yeast and mice. This finding suggests a model in which the orientation of adjacent Spo11 molecules is fixed relative to the DNA helix-a proposal supported by the in vitro DNA-binding properties of the Spo11 core complex. Deep sequencing of meiotic progeny identifies recombination scars that are consistent with repair initiated from gaps generated by adjacent Spo11 DSBs. Collectively, these results revise our present understanding of the mechanics of Spo11-DSB formation and expand on the original concepts of gap repair during meiosis to include DNA gaps that are generated by Spo11 itself.
Asunto(s)
Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/genética , Meiosis , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Animales , Reparación del ADN , Ratones , Ratones NoqueadosRESUMEN
The separation of germ cell populations from the soma is part of the evolutionary transition to multicellularity. Only genetic information present in the germ cells will be inherited by future generations, and any molecular processes affecting the germline genome are therefore likely to be passed on. Despite its prevalence across taxonomic kingdoms, we are only starting to understand details of the underlying micro-evolutionary processes occurring at the germline genome level. These include segregation, recombination, mutation and selection and can occur at any stage during germline differentiation and mitotic germline proliferation to meiosis and post-meiotic gamete maturation. Selection acting on germ cells at any stage from the diploid germ cell to the haploid gametes may cause significant deviations from Mendelian inheritance and may be more widespread than previously assumed. The mechanisms that affect and potentially alter the genomic sequence and allele frequencies in the germline are pivotal to our understanding of heritability. With the rise of new sequencing technologies, we are now able to address some of these unanswered questions. In this review, we comment on the most recent developments in this field and identify current gaps in our knowledge.
Asunto(s)
Células Germinativas , Meiosis , Evolución Biológica , Genoma , Meiosis/genética , MutaciónRESUMEN
The three-dimensional architecture of the genome governs its maintenance, expression and transmission. The cohesin protein complex organizes the genome by topologically linking distant loci, and is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres1-6. Here we report the three-dimensional structure of pericentromeres in budding yeast (Saccharomyces cerevisiae) and establish the relationship between genome organization and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin, and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organized into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Reorienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation during mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading shapes pericentromeres into a structure that is competent for chromosome segregation. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded, as well as the restructuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between the three-dimensional genome organization of a specific chromosomal domain and cellular function.
Asunto(s)
Centrómero/genética , Centrómero/metabolismo , Genes Fúngicos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/metabolismo , Centrómero/química , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Genoma Fúngico/genética , Viabilidad Microbiana/genética , Mitosis/genética , Conformación Molecular , CohesinasRESUMEN
During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we use Saccharomyces cerevisiae to explore how this elaborate three-dimensional chromosome organisation is linked to genomic sequence. As cells enter meiosis, we observe that strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion with growth limited by barriers, in which a heterogeneous population of expanding loops develop along the chromosome. Importantly, CTCF, the factor that imposes similar features in mammalian interphase, is absent in S. cerevisiae, suggesting alternative mechanisms of barrier formation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process.
Asunto(s)
Cromosomas Fúngicos/fisiología , Meiosis , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/química , Simulación por Computador , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Sinaptonémico/metabolismo , CohesinasRESUMEN
DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes-and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage-distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.
Asunto(s)
Reparación del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Antineoplásicos Fitogénicos/farmacología , Secuencia de Bases , Factor de Unión a CCCTC/genética , ADN/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Cadena Simple/efectos de los fármacos , Etopósido/farmacología , Humanos , Mapeo NucleótidoRESUMEN
Meiosis is a specialized two-step cell division responsible for genome haploidization and the generation of genetic diversity during gametogenesis. An integral and distinctive feature of the meiotic program is the evolutionarily conserved initiation of homologous recombination (HR) by the developmentally programmed induction of DNA double-strand breaks (DSBs). The inherently dangerous but essential act of DSB formation is subject to multiple forms of stringent and self-corrective regulation that collectively ensure fruitful and appropriate levels of genetic exchange without risk to cellular survival. Within this article we focus upon an emerging element of this control--spatial regulation--detailing recent advances made in understanding how DSBs are evenly distributed across the genome, and present a unified view of the underlying patterning mechanisms employed.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Meiosis/fisiología , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Recombinación Homóloga/fisiología , Humanos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Meiotic recombination is a critical step in gametogenesis for many organisms, enabling the creation of genetically diverse haploid gametes. In each meiotic cell, recombination is initiated by numerous DNA double-strand breaks (DSBs) created by Spo11, the evolutionarily conserved topoisomerase-like protein, but how these DSBs are distributed relatively uniformly across the four chromatids that make up each chromosome pair is poorly understood. Here we employ Saccharomyces cerevisiae to demonstrate distance-dependent DSB interference in cis (in which the occurrence of a DSB suppresses adjacent DSB formation)--a process that is mediated by the conserved DNA damage response kinase, Tel1(ATM). The inhibitory function of Tel1 acts on a relatively local scale, while over large distances DSBs have a tendency to form independently of one another even in the presence of Tel1. Notably, over very short distances, loss of Tel1 activity causes DSBs to cluster within discrete zones of concerted DSB activity. Our observations support a hierarchical view of recombination initiation where Tel1(ATM) prevents clusters of DSBs, and further suppresses DSBs within the surrounding chromosomal region. Such collective negative regulation will help to ensure that recombination events are dispersed evenly and arranged optimally for genetic exchange and efficient chromosome segregation.
Asunto(s)
Roturas del ADN de Doble Cadena , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Meiosis/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , 3-Isopropilmalato Deshidrogenasa/genética , Oxidorreductasas de Alcohol/genética , Aminohidrolasas/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cromosomas Fúngicos/genética , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/metabolismo , Genes Fúngicos/genética , Recombinación Homóloga/genética , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Pirofosfatasas/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Roturas del ADN de Doble Cadena , Homeostasis/fisiología , Meiosis/fisiología , Animales , Ciclo Celular/fisiología , HumanosRESUMEN
MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/química , Fase G2 , Reparación del ADN por Recombinación , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Rayos gamma/efectos adversos , Humanos , Proteína Homóloga de MRE11 , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismoRESUMEN
During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.
Asunto(s)
Roturas del ADN de Doble Cadena , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Meiosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Reparación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismoRESUMEN
Repair of DNA double-strand breaks (DSBs) by homologous recombination requires resection of 5'-termini to generate 3'-single-strand DNA tails. Key components of this reaction are exonuclease 1 and the bifunctional endo/exonuclease, Mre11 (refs 2-4). Mre11 endonuclease activity is critical when DSB termini are blocked by bound protein--such as by the DNA end-joining complex, topoisomerases or the meiotic transesterase Spo11 (refs 7-13)--but a specific function for the Mre11 3'-5' exonuclease activity has remained elusive. Here we use Saccharomyces cerevisiae to reveal a role for the Mre11 exonuclease during the resection of Spo11-linked 5'-DNA termini in vivo. We show that the residual resection observed in Exo1-mutant cells is dependent on Mre11, and that both exonuclease activities are required for efficient DSB repair. Previous work has indicated that resection traverses unidirectionally. Using a combination of physical assays for 5'-end processing, our results indicate an alternative mechanism involving bidirectional resection. First, Mre11 nicks the strand to be resected up to 300 nucleotides from the 5'-terminus of the DSB--much further away than previously assumed. Second, this nick enables resection in a bidirectional manner, using Exo1 in the 5'-3' direction away from the DSB, and Mre11 in the 3'-5' direction towards the DSB end. Mre11 exonuclease activity also confers resistance to DNA damage in cycling cells, suggesting that Mre11-catalysed resection may be a general feature of various DNA repair pathways.