RESUMEN
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
In most eukaryotes, meiotic crossovers (COs) are limited to 1-3 per chromosome, and are prevented from occurring close to one another by CO interference. The fission yeast Schizosaccharomyces pombe, an exception to these general rules, was reported to have the highest CO number per chromosome and no or weak interference. However, global CO frequency was indirectly estimated, calling for confirmation. Here, we used an innovative strategy to determine COs genome-wide in S. pombe. We confirmed weak CO interference, acting at physical distances compatible with the patterning of recombination precursors. We revealed a slight co-variation in CO number between chromosomes, suggesting that a limiting pro-CO factor varies between meiocytes. CO number per chromosome varies proportionally with chromosome size, with the three chromosomes having, on average, 15.9, 12.5, and 7.0 COs, respectively. This reinforces S. pombe's status as the eukaryote with the highest CO number per chromosome described to date.
RESUMEN
Meiotic recombination is a driving force for genome evolution, deeply characterized in a few model species, notably in the budding yeast Saccharomyces cerevisiae. Interestingly, Zip2, Zip3, Zip4, Spo16, Msh4, and Msh5, members of the so-called ZMM pathway that implements the interfering meiotic crossover pathway in S. cerevisiae, have been lost in Lachancea yeast species after the divergence of Lachancea kluyveri from the rest of the clade. In this context, after investigating meiosis in L. kluyveri, we determined the meiotic recombination landscape of Lachancea waltii. Attempts to generate diploid strains with fully hybrid genomes invariably resulted in strains with frequent whole-chromosome aneuploidy and multiple extended regions of loss of heterozygosity (LOH), which mechanistic origin is so far unclear. Despite the lack of multiple ZMM pro-crossover factors in L. waltii, numbers of crossovers and noncrossovers per meiosis were higher than in L. kluyveri but lower than in S. cerevisiae, for comparable genome sizes. Similar to L. kluyveri but opposite to S. cerevisiae, L. waltii exhibits an elevated frequency of zero-crossover bivalents. Lengths of gene conversion tracts for both crossovers and non-crossovers in L. waltii were comparable to those observed in S. cerevisiae and shorter than in L. kluyveri despite the lack of Mlh2, a factor limiting conversion tract size in S. cerevisiae. L. waltii recombination hotspots were not shared with either S. cerevisiae or L. kluyveri, showing that meiotic recombination hotspots can evolve at a rather limited evolutionary scale within budding yeasts. Finally, L. waltii crossover interference was reduced relative to S. cerevisiae, with interference being detected only in the 25 kb distance range. Detection of positive inference only at short distance scales in the absence of multiple ZMM factors required for interference-sensitive crossovers in other systems likely reflects interference between early recombination precursors such as DSBs.
Asunto(s)
Meiosis , Intercambio Genético , Proteínas de Unión al ADN/genética , Meiosis/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas MutL/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Break-induced replication (BIR) is a highly mutagenic eukaryotic homologous DNA recombination pathway that repairs one-ended DNA double strand breaks such as broken DNA replication forks and eroded telomeres. While searching for cis-acting factors regulating ectopic BIR efficiency, we found that ectopic BIR efficiency is the highest close to chromosome ends. The variations of ectopic BIR efficiency as a function of the length of DNA to replicate can be described as a combination of two decreasing exponential functions, a property in line with repeated cycles of strand invasion, elongation and dissociation that characterize BIR. Interestingly, the apparent processivity of ectopic BIR depends on the length of DNA already synthesized. Ectopic BIR is more susceptible to disruption during the synthesis of the first ~35-40 kb of DNA than later, notably when the template chromatid is being transcribed or heterochromatic. Finally, we show that the Srs2 helicase promotes ectopic BIR from both telomere proximal and telomere distal regions in diploid cells but only from telomere proximal sites in haploid cells. Altogether, we bring new light on the factors impacting a last resort DNA repair pathway.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Transactivadores , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Replicación del ADN/genética , Recombinación Homóloga , Proteínas de Saccharomyces cerevisiae/genética , Transactivadores/genéticaRESUMEN
Meiotic recombination is an essential biological process that ensures faithful chromosome segregation and promotes parental allele shuffling. Tetrad analysis is a powerful approach to quantify the genetic makeups and recombination landscapes of meiotic products. Here we present RecombineX (https://github.com/yjx1217/RecombineX), a generalized computational framework that automates the full workflow of marker identification, gamete genotyping, and tetrad-based recombination profiling based on any organism or genetic background with batch processing capability. Aside from conventional reference-based analysis, RecombineX can also perform analysis based on parental genome assemblies, which facilitates analyzing meiotic recombination landscapes in their native genomic contexts. Additional features such as copy number variation profiling and missing genotype inference further enhance downstream analysis. RecombineX also includes a dedicate module for simulating the genomes and reads of recombinant tetrads, which enables fine-tuned simulation-based hypothesis testing. This simulation module revealed the power and accuracy of RecombineX even when analyzing tetrads with very low sequencing depths (e.g., 1-2X). Tetrad sequencing data from the budding yeast Saccharomyces cerevisiae and green alga Chlamydomonas reinhardtii were further used to demonstrate the accuracy and robustness of RecombineX for organisms with both small and large genomes, manifesting RecombineX as an all-around one stop solution for future tetrad analysis. Interestingly, our re-analysis of the budding yeast tetrad sequencing data with RecombineX and Oxford Nanopore sequencing revealed two unusual structural rearrangement events that were not noticed before, which exemplify the occasional genome instability triggered by meiosis.
Asunto(s)
Variaciones en el Número de Copia de ADN , Meiosis , Genotipo , Células Germinativas , Recombinación Homóloga , Meiosis/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Dissecting the genetic basis of complex trait remains a real challenge. The budding yeast Saccharomyces cerevisiae has become a model organism for studying quantitative traits, successfully increasing our knowledge in many aspects. However, the exploration of the genotype-phenotype relationship in non-model yeast species could provide a deeper insight into the genetic basis of complex traits. Here, we have studied this relationship in the Lachancea waltii species which diverged from the S. cerevisiae lineage prior to the whole-genome duplication. By performing linkage mapping analyses in this species, we identified 86 quantitative trait loci (QTL) impacting the growth in a large number of conditions. The distribution of these loci across the genome has revealed two major QTL hotspots. A first hotspot corresponds to a general growth QTL, impacting a wide range of conditions. By contrast, the second hotspot highlighted a trade-off with a disadvantageous allele for drug-free conditions which proved to be advantageous in the presence of several drugs. Finally, a comparison of the detected QTL in L. waltii with those which had been previously identified for the same trait in a closely related species, Lachancea kluyveri was performed. This analysis clearly showed the absence of shared QTL across these species. Altogether, our results represent a first step toward the exploration of the genetic architecture of quantitative trait across different yeast species.
Asunto(s)
Sitios de Carácter Cuantitativo , Saccharomycetales , Mapeo Cromosómico , Disección , Ligamiento Genético , Genotipo , Fenotipo , Filogenia , Saccharomyces cerevisiae/genética , Saccharomycetales/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
Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLß heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLß recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLß ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLß may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1's activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLß ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.
Asunto(s)
ADN Helicasas/metabolismo , Conversión Génica , Recombinación Homóloga , Meiosis/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Roturas del ADN de Doble Cadena , ADN Helicasas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Espectrometría de Masas , Proteínas MutL/genética , Proteínas MutL/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Recombinantes , Proteína de Replicación C/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The budding yeast, Saccharomyces cerevisiae, has served as a model for nearly a century to understand the principles of the eukaryotic life cycle. The canonical life cycle of S. cerevisiae comprises a regular alternation between haploid and diploid phases. Haploid gametes generated by sporulation are expected to quickly restore the diploid phase mainly through inbreeding via intratetrad mating or haploselfing, thereby promoting genome homozygotization. However, recent large population genomics data unveiled that heterozygosity and polyploidy are unexpectedly common. This raises the interesting paradox of a haplo-diplobiontic species being well-adapted to inbreeding and able to maintain high levels of heterozygosity and polyploidy, thereby suggesting an unanticipated complexity of the yeast life cycle. Here, we propose that unprogrammed mating type switching, heterothallism, reduced spore formation and viability, cell-cell fusion and dioecy could play key and uncharted contributions to generate and maintain heterozygosity through polyploidization.
Asunto(s)
Genes del Tipo Sexual de los Hongos , Genoma Fúngico , Heterocigoto , Saccharomycetales/crecimiento & desarrollo , Saccharomycetales/genética , Estadios del Ciclo de Vida/genética , Estadios del Ciclo de Vida/fisiología , Reproducción , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Saccharomycetales/clasificación , Saccharomycetales/fisiologíaRESUMEN
In chromosome conformation capture experiments (Hi-C), the accuracy with which contacts are detected varies due to the uneven distribution of restriction sites along genomes. In addition, repeated sequences or homologous regions remain indistinguishable because of the ambiguities they introduce during the alignment of the sequencing reads. We addressed both limitations by designing and engineering 144 kb of a yeast chromosome with regularly spaced restriction sites (Syn-HiC design). In the Syn-HiC region, Hi-C signal-to-noise ratio is enhanced and can be used to measure the shape of an unbiased distribution of contact frequencies, allowing to propose a robust definition of a Hi-C experiment resolution. The redesigned region is also distinguishable from its native homologous counterpart in an otherwise isogenic diploid strain. As a proof of principle, we tracked homologous chromosomes during meiotic prophase in synchronized and pachytene-arrested cells and captured important features of their spatial reorganization, such as chromatin restructuration into arrays of Rec8-delimited loops, centromere declustering, individualization, and pairing. Overall, we illustrate the promises held by redesigning genomic regions to explore complex biological questions.
Asunto(s)
Cromosomas Fúngicos/genética , Schizosaccharomyces/fisiología , Tamaño del Genoma , Meiosis , Schizosaccharomyces/genética , Biología de Sistemas/métodosRESUMEN
Meiotic recombination is essential for fertility and allelic shuffling. Canonical recombination models fail to capture the observed complexity of meiotic recombinants. Here, by combining genome-wide meiotic heteroduplex DNA patterns with meiotic DNA double-strand break (DSB) sites, we show that part of this complexity results from frequent template switching during synthesis-dependent strand annealing that yields noncrossovers and from branch migration of double Holliday junction (dHJ)-containing intermediates that mainly yield crossovers. This complexity also results from asymmetric positioning of crossover intermediates relative to the initiating DSB and Msh2-independent conversions promoted by the suspected dHJ resolvase Mlh1-3 as well as Exo1 and Sgs1. Finally, we show that dHJ resolution is biased toward cleavage of the pair of strands containing newly synthesized DNA near the junctions and that this bias can be decoupled from the crossover-biased dHJ resolution. These properties are likely conserved in eukaryotes containing ZMM proteins, which includes mammals.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Cruciforme , ADN de Hongos/genética , Meiosis , Ácidos Nucleicos Heterodúplex/genética , Recombinación Genética , Saccharomyces cerevisiae/genética , ADN de Hongos/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Meiotic recombination is a major factor of genome evolution, deeply characterized in only a few model species, notably the yeast Saccharomyces cerevisiae. Consequently, little is known about variations of its properties across species. In this respect, we explored the recombination landscape of Lachancea kluyveri, a protoploid yeast species that diverged from the Saccharomyces genus more than 100 million years ago and we found striking differences with S. cerevisiae. These variations include a lower recombination rate, a higher frequency of chromosomes segregating without any crossover and the absence of recombination on the chromosome arm containing the sex locus. In addition, although well conserved within the Saccharomyces clade, the S. cerevisiae recombination hotspots are not conserved over a broader evolutionary distance. Finally and strikingly, we found evidence of frequent reversal of commitment to meiosis, resulting in return to mitotic growth after allele shuffling. Identification of this major but underestimated evolutionary phenomenon illustrates the relevance of exploring non-model species.
Asunto(s)
Genoma Fúngico , Recombinación Homóloga , Meiosis/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Cromosomas Fúngicos/genética , ADN de Hongos/genética , Evolución Molecular , Mitosis/genética , Filogenia , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/clasificación , Análisis de Secuencia de ADNRESUMEN
Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLß complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLß preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLß is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.
Asunto(s)
ADN Helicasas/metabolismo , Conversión Génica , Homólogo 1 de la Proteína MutL/metabolismo , Proteínas MutL/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Cromosomas Fúngicos/metabolismoRESUMEN
Since more than a decade ago, Saccharomyces cerevisiae has been used as a model to dissect complex traits, revealing the genetic basis of a large number of traits in fine detail. However, to have a more global view of the genetic architecture of traits across species, the examination of the molecular basis of phenotypes within non-conventional species would undoubtedly be valuable. In this respect, the Saccharomycotina yeasts represent ideal and potential non-model organisms. Here we sought to assess the feasibility of genetic mapping by bulk segregant analysis in the protoploid Lachancea kluyveri (formerly S. kluyveri) yeast species, a distantly related species to S. cerevisiae For this purpose, we designed a fluorescent mating-type marker, compatible with any mating-competent strains representative of this species, to rapidly create a large population of haploid segregants (>10(5) cells). Quantitative trait loci can be mapped by selecting and sequencing an enriched pool of progeny with extreme phenotypic values. As a test bed, we applied this strategy and mapped the causal loci underlying halotolerance phenotypes in L. kluyveri Overall, this study demonstrates that bulk segregant mapping is a powerful way for investigating the genetic basis of natural variations in non-model yeast organisms and more precisely in L. kluyveri.
Asunto(s)
Mapeo Cromosómico/métodos , Sitios de Carácter Cuantitativo , Saccharomycetales/genética , Genes Reporteros , Coloración y EtiquetadoRESUMEN
Reconstructing genome history is complex but necessary to reveal quantitative principles governing genome evolution. Such reconstruction requires recapitulating into a single evolutionary framework the evolution of genome architecture and gene repertoire. Here, we reconstructed the genome history of the genus Lachancea that appeared to cover a continuous evolutionary range from closely related to more diverged yeast species. Our approach integrated the generation of a high-quality genome data set; the development of AnChro, a new algorithm for reconstructing ancestral genome architecture; and a comprehensive analysis of gene repertoire evolution. We found that the ancestral genome of the genus Lachancea contained eight chromosomes and about 5173 protein-coding genes. Moreover, we characterized 24 horizontal gene transfers and 159 putative gene creation events that punctuated species diversification. We retraced all chromosomal rearrangements, including gene losses, gene duplications, chromosomal inversions and translocations at single gene resolution. Gene duplications outnumbered losses and balanced rearrangements with 1503, 929, and 423 events, respectively. Gene content variations between extant species are mainly driven by differential gene losses, while gene duplications remained globally constant in all lineages. Remarkably, we discovered that balanced chromosomal rearrangements could be responsible for up to 14% of all gene losses by disrupting genes at their breakpoints. Finally, we found that nonsynonymous substitutions reached fixation at a coordinated pace with chromosomal inversions, translocations, and duplications, but not deletions. Overall, we provide a granular view of genome evolution within an entire eukaryotic genus, linking gene content, chromosome rearrangements, and protein divergence into a single evolutionary framework.
Asunto(s)
Ascomicetos/genética , Cromosomas Fúngicos/genética , Evolución Molecular , Reordenamiento Génico , Genoma Fúngico , Modelos Genéticos , FilogeniaRESUMEN
Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. Although CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favour HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In summary, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Fosforilación/fisiología , Western Blotting , Línea Celular , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/genética , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Reparación del ADN/fisiología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Fosforilación/genéticaRESUMEN
Structure-specific DNA endonucleases have critical roles during DNA replication, repair and recombination, yet they also have the potential for causing genome instability. Controlling these enzymes may be essential to ensure efficient processing of ad hoc substrates and to prevent random, unscheduled processing of other DNA structures, but it is unknown whether structure-specific endonucleases are regulated in response to DNA damage. Here, we uncover DNA damage-induced activation of Mus81-Eme1 Holliday junction resolvase in fission yeast. This new regulation requires both Cdc2(CDK1)- and Rad3(ATR)-dependent phosphorylation of Eme1. Mus81-Eme1 activation prevents gross chromosomal rearrangements in cells lacking the BLM-related DNA helicase Rqh1. We propose that linking Mus81-Eme1 DNA damage-induced activation to cell-cycle progression ensures efficient resolution of Holliday junctions that escape dissolution by Rqh1-TopIII while preventing unnecessary DNA cleavages.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica , Resolvasas de Unión Holliday/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Modelos Biológicos , Fosforilación , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5'-3' degradation of DSB ends that yields the 3' single-stranded DNA required for the loading of checkpoint and recombination proteins. In yeast, the Mre11-Rad50-Xrs2 complex (Xrs2 is known as NBN or NBS1 in humans) and Sae2 (known as RBBP8 or CTIP in humans) initiate end resection, whereas long-range resection depends on the exonuclease Exo1, or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 together with the endonuclease Dna2 (refs 1-6). DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood. Here we show that the yeast Saccharomyces cerevisiae Fun30 protein and its human counterpart SMARCAD1 (ref. 8), two poorly characterized ATP-dependent chromatin remodellers of the Snf2 ATPase family, are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates the repair of camptothecin-induced DNA lesions, although it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs, and the kinetics of recruitment is similar to that of EXO1. The loss of SMARCAD1 impairs end resection and recombinational DNA repair, and renders cells hypersensitive to DNA damage resulting from camptothecin or poly(ADP-ribose) polymerase inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodellers in controlling end resection, homologous recombination and genome stability in the context of chromatin.
Asunto(s)
Ensamble y Desensamble de Cromatina , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Reparación del ADN , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Camptotecina/farmacología , Línea Celular , Supervivencia Celular , ADN/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , ADN Helicasas/deficiencia , ADN Helicasas/genética , Reparación del ADN/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Inestabilidad Genómica/genética , Histonas/metabolismo , Recombinación Homóloga/genética , Humanos , Mutación , Nucleosomas/genética , Nucleosomas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , RecQ Helicasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.