RESUMEN
Centromeres are essential for chromosome segregation in most animals and plants yet are among the most rapidly evolving genome elements. The mechanisms underlying this paradoxical phenomenon remain enigmatic. Here, we report that human centromeres innately harbor a striking enrichment of DNA breaks within functionally active centromere regions. Establishing a single-cell imaging strategy that enables comparative assessment of DNA breaks at repetitive regions, we show that centromeric DNA breaks are induced not only during active cellular proliferation but also de novo during quiescence. Markedly, centromere DNA breaks in quiescent cells are resolved enzymatically by the evolutionarily conserved RAD51 recombinase, which in turn safeguards the specification of functional centromeres. This study highlights the innate fragility of centromeres, which may have been co-opted over time to reinforce centromere specification while driving rapid evolution. The findings also provide insights into how fragile centromeres are likely to contribute to human disease.
Asunto(s)
Centrómero , ADN , Animales , Humanos , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Recombinación GenéticaRESUMEN
Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.
Asunto(s)
Roturas del ADN de Cadena Simple , ADN Helicasas/metabolismo , Reparación del ADN , Replicación del ADN , ADN de Hongos/metabolismo , ADN de Cadena Simple/metabolismo , Mutación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sitios de Unión , Cromosomas Fúngicos , ADN Helicasas/genética , ADN de Hongos/genética , ADN de Cadena Simple/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Mitosis , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Reparación del ADN por Recombinación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaciónRESUMEN
Detecting DNA breaks in defined regions of the genome is critical to advancing our understanding of genome stability maintenance. Here, we present exo-FISH, a protocol to label exposed single-stranded DNA in defined repetitive regions of mammalian genomes by combining in vitro restriction enzyme digestion on fixed cells with fluorescence in situ hybridization (FISH). We describe steps for cell harvesting and fixation, slide treatments, and FISH probe hybridization. We then detail procedures for imaging and analysis. For complete details on the use and execution of this protocol, please refer to Saayman et al. (2023).1.
Asunto(s)
ADN , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Hibridación Fluorescente in Situ/métodos , ADN/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , ADN de Cadena Simple , Roturas del ADN , Mamíferos/genéticaRESUMEN
DNA double-strand breaks (DSBs) can result from both exogenous and endogenous sources and are potentially toxic lesions to the human genome. If improperly repaired, DSBs can threaten genome integrity and contribute to premature ageing, neurodegenerative disorders and carcinogenesis. Through decades of work on genome stability, it has become evident that certain regions of the genome are inherently more prone to breakage than others, known as genome instability hotspots. Recent advancements in sequencing-based technologies now enable the profiling of genome-wide distributions of DSBs, also known as breakomes, to systematically map these instability hotspots. Here, we review the application of these technologies and their implications for our current understanding of the genomic regions most likely to drive genome instability. These breakomes ultimately highlight both new and established breakage hotspots including actively transcribed regions, loop boundaries and early-replicating regions of the genome. Further, these breakomes challenge the paradigm that DNA breakage primarily occurs in hard-to-replicate regions. With these advancements, we begin to gain insights into the biological mechanisms both invoking and protecting against genome instability.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Animales , ADN/genética , Reparación del ADN/genética , Genoma Humano , Inestabilidad Genómica , Humanos , Mamíferos/genéticaRESUMEN
The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.
Asunto(s)
Anafase/genética , Cromatina/metabolismo , Reparación del ADN , Puntos de Control de la Fase M del Ciclo Celular/genética , Recombinasa Rad51/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Segregación Cromosómica , ADN/biosíntesis , Daño del ADN , Replicación del ADN , Inestabilidad Genómica , Humanos , Microscopía Intravital , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Quinasa Tipo Polo 1RESUMEN
Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.