RESUMEN
In Saccharomyces cerevisiae, dicentric chromosomes stemming from telomere fusions preferentially break at the fusion. This process restores a normal karyotype and protects chromosomes from the detrimental consequences of accidental fusions. Here, we address the molecular basis of this rescue pathway. We observe that tandem arrays tightly bound by the telomere factor Rap1 or a heterologous high-affinity DNA binding factor are sufficient to establish breakage hotspots, mimicking telomere fusions within dicentrics. We also show that condensins generate forces sufficient to rapidly refold dicentrics prior to breakage by cytokinesis and are essential to the preferential breakage at telomere fusions. Thus, the rescue of fused telomeres results from a condensin- and Rap1-driven chromosome folding that favors fusion entrapment where abscission takes place. Because a close spacing between the DNA-bound Rap1 molecules is essential to this process, Rap1 may act by stalling condensins.
Asunto(s)
Adenosina Trifosfatasas/genética , Cromosomas Fúngicos/metabolismo , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Telómero/metabolismo , Factores de Transcripción/genética , Adenosina Trifosfatasas/metabolismo , Puntos de Rotura del Cromosoma , Cromosomas Fúngicos/ultraestructura , Citocinesis/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Cariotipo , Modelos Genéticos , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Telómero/ultraestructura , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Dicentric chromosomes are unstable products of erroneous DNA repair events that can lead to further genome rearrangements and extended gene copy number variations. During mitosis, they form anaphase bridges, resulting in chromosome breakage by an unknown mechanism. In budding yeast, dicentrics generated by telomere fusion break at the fusion, a process that restores the parental karyotype and protects cells from rare accidental telomere fusion. Here, we observed that dicentrics lacking telomere fusion preferentially break within a 25- to 30-kb-long region next to the centromeres. In all cases, dicentric breakage requires anaphase exit, ruling out stretching by the elongated mitotic spindle as the cause of breakage. Instead, breakage requires cytokinesis. In the presence of dicentrics, the cytokinetic septa pinch the nucleus, suggesting that dicentrics are severed after actomyosin ring contraction. At this time, centromeres and spindle pole bodies relocate to the bud neck, explaining how cytokinesis can sever dicentrics near centromeres.
Asunto(s)
Centrómero/genética , Rotura Cromosómica , Cromosomas Fúngicos/genética , Citocinesis , Saccharomyces cerevisiae/genética , Telómero/metabolismo , División del Núcleo Celular , MitosisRESUMEN
Replication of vertebrate genomes is tightly regulated to ensure accurate duplication, but our understanding of the interplay between genetic and epigenetic factors in this regulation remains incomplete. Here, we investigated the involvement of three elements enriched at gene promoters and replication origins: guanine-rich motifs potentially forming G-quadruplexes (pG4s), nucleosome-free regions (NFRs), and the histone variant H2A.Z, in the firing of origins of replication in vertebrates. We show that two pG4s on the same DNA strand (dimeric pG4s) are sufficient to induce the assembly of an efficient minimal replication origin without inducing transcription in avian DT40 cells. Dimeric pG4s in replication origins are associated with formation of an NFR next to precisely-positioned nucleosomes enriched in H2A.Z on this minimal origin and genome-wide. Thus, our data suggest that dimeric pG4s are important for the organization and duplication of vertebrate genomes. It supports the hypothesis that a nucleosome close to an NFR is a shared signal for the formation of replication origins in eukaryotes.
Asunto(s)
G-Cuádruplex , Nucleosomas , Animales , Nucleosomas/genética , Origen de Réplica/genética , Replicación del ADN/genética , Histonas/genética , Histonas/metabolismo , Vertebrados/genética , Vertebrados/metabolismoRESUMEN
The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar "recombination compartment" where AID could be sequestered thus limiting the extent of its potentially deleterious off-target effects.