RESUMO
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.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Cromossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Recombinação Homóloga/genética , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In the yeast Saccharomyces cerevisiae, the silent information regulator (SIR) complex assembles heterochromatin-like chromatin at sub-telomeric chromosomal regions. SIR-mediated repressive chromatin limits DNA double-strand break (DSB) resection, thus protecting damaged chromosome ends during homologous recombination (HR). As resection initiation represents the crossroads between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR-mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Via physical interaction with the Sae2 protein, Sir3 impairs Sae2-dependent functions of the MRX (Mre11-Rad50-Xrs2) complex, thereby limiting Mre11-mediated resection, delaying MRX removal from DSB ends, and promoting NHEJ.
Assuntos
Reparo do DNA por Junção de Extremidades , Endonucleases/metabolismo , Heterocromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Endonucleases/química , Mutação Puntual/genética , Ligação Proteica , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/química , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Telômero/metabolismoRESUMO
Homologous recombination (HR) is a conserved mechanism that repairs broken chromosomes via intact homologous sequences. How different genomic, chromatin and subnuclear contexts influence HR efficiency and outcome is poorly understood. We developed an assay to assess HR outcome by gene conversion (GC) and break-induced replication (BIR), and discovered that subtelomeric double-stranded breaks (DSBs) are preferentially repaired by BIR despite the presence of flanking homologous sequences. Overexpression of a silencing-deficient SIR3 mutant led to active grouping of telomeres and specifically increased the GC efficiency between subtelomeres. Thus, physical distance limits GC at subtelomeres. However, the repair efficiency between reciprocal intrachromosomal and subtelomeric sequences varies up to 15-fold, depending on the location of the DSB, indicating that spatial proximity is not the only limiting factor for HR EXO1 deletion limited the resection at subtelomeric DSBs and improved GC efficiency. The presence of repressive chromatin at subtelomeric DSBs also favoured recombination, by counteracting EXO1-mediated resection. Thus, repressive chromatin promotes HR at subtelomeric DSBs by limiting DSB resection and protecting against genetic information loss.
Assuntos
Cromatina/genética , Quebras de DNA de Cadeia Dupla , Recombinação Genética , Telômero/genética , DNA Fúngico/genética , Leveduras/genéticaRESUMO
Genomic DNA is constantly exposed to damage. Among the lesion in DNA, double-strand breaks (DSB), because they disrupt the two strands of the DNA double helix, are the more dangerous. DSB are repaired through two evolutionary conserved mechanisms: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Whereas NHEJ simply reseals the double helix with no or minimal processing, HR necessitates the formation of a 3'ssDNA through the processing of DSB ends by the resection machinery and relies on the recognition and pairing of this 3'ssDNA tails with an intact homologous sequence. Despite years of active research on HR, the manner by which the two homologous sequences find each other in the crowded nucleus, and how this modulates HR efficiency, only recently emerges. Here, we review recent advances in our understanding of the factors limiting the search of a homologous sequence during HR.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Replicação do DNA , Recombinação Homóloga , Animais , DNA/genética , DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos GenéticosRESUMO
Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not only required for sister chromatid cohesion but also plays a major role determining the structure of individual chromatids in metaphase. Separase cleavage is thought to remove all cohesin complexes from chromosomes to initiate anaphase. It is thus not clear how the length and organisation of segregating chromatids is maintained during anaphase in the absence of cohesin. Here, we show that degradation of cohesin at the anaphase onset causes aberrant chromatid segregation. Hi-C analysis on segregating chromatids demonstrates that cohesin depletion causes loss of intrachromatid organisation. Surprisingly, tobacco etch virus (TEV)-mediated cleavage of cohesin does not dramatically disrupt chromatid organisation in anaphase, explaining why bulk segregation is achieved. In addition, we identified a small pool of cohesin complexes bound to telophase chromosomes in wild-type cells and show that they play a role in the organisation of centromeric regions. Our data demonstrates that in yeast cells cohesin function is not over in metaphase, but extends to the anaphase period when chromatids are segregating.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Saccharomyces cerevisiae , Anáfase/genética , Cromátides , Cromatina/química , Cromatina/metabolismo , Saccharomyces cerevisiae/genética , Separase/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
Homologous recombination repairs DNA double-strand breaks (DSB) using an intact dsDNA molecule as a template. It entails a homology search step, carried out along a conserved RecA/Rad51-ssDNA filament assembled on each DSB end. Whether, how and to what extent a DSB impacts chromatin folding, and how this (re)organization in turns influences the homology search process, remain ill-defined. Here we characterize two layers of spatial chromatin reorganization following DSB formation in Saccharomyces cerevisiae. Although cohesin folds chromosomes into cohesive arrays of ~20-kb-long chromatin loops as cells arrest in G2/M, the DSB-flanking regions interact locally in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin, Mec1ATR, Rad52 and Rad51. This local structure blocks cohesin progression, constraining the DSB region at the base of a loop. Functionally, cohesin promotes DSB-dsDNA interactions and donor identification in cis, while inhibiting them in trans. This study identifies multiple direct and indirect ways by which cohesin regulates homology search during recombinational DNA repair.