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1.
Nature ; 623(7985): 183-192, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37853125

RESUMO

The DNA damage response is essential to safeguard genome integrity. Although the contribution of chromatin in DNA repair has been investigated1,2, the contribution of chromosome folding to these processes remains unclear3. Here we report that, after the production of double-stranded breaks (DSBs) in mammalian cells, ATM drives the formation of a new chromatin compartment (D compartment) through the clustering of damaged topologically associating domains, decorated with γH2AX and 53BP1. This compartment forms by a mechanism that is consistent with polymer-polymer phase separation rather than liquid-liquid phase separation. The D compartment arises mostly in G1 phase, is independent of cohesin and is enhanced after pharmacological inhibition of DNA-dependent protein kinase (DNA-PK) or R-loop accumulation. Importantly, R-loop-enriched DNA-damage-responsive genes physically localize to the D compartment, and this contributes to their optimal activation, providing a function for DSB clustering in the DNA damage response. However, DSB-induced chromosome reorganization comes at the expense of an increased rate of translocations, also observed in cancer genomes. Overall, we characterize how DSB-induced compartmentalization orchestrates the DNA damage response and highlight the critical impact of chromosome architecture in genomic instability.


Assuntos
Compartimento Celular , Cromatina , Dano ao DNA , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Fase G1 , Histonas/metabolismo , Neoplasias/genética , Estruturas R-Loop , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
2.
Nature ; 590(7847): 660-665, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33597753

RESUMO

The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genome integrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers the establishment of megabase-sized, chromatin domains decorated with phosphorylated histone H2AX (γH2AX), which act as seeds for the formation of DNA-damage response foci1. It is unclear how these foci are rapidly assembled to establish a 'repair-prone' environment within the nucleus. Topologically associating domains are a key feature of 3D genome organization that compartmentalize transcription and replication, but little is known about their contribution to DNA repair processes2,3. Here we show that topologically associating domains are functional units of the DNA damage response, and are instrumental for the correct establishment of γH2AX-53BP1 chromatin domains in a manner that involves one-sided cohesin-mediated loop extrusion on both sides of the DSB. We propose a model in which H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. Our work highlights the importance of chromosome conformation in the maintenance of genome integrity and demonstrates the establishment of a chromatin modification by loop extrusion.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/química , DNA/metabolismo , Conformação de Ácido Nucleico , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , Genoma/genética , Histonas/metabolismo , Humanos , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Fosforilação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Coesinas
3.
Genes Dev ; 33(17-18): 1175-1190, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31395742

RESUMO

The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. DNA double-strand breaks (DSBs) within rDNA induce both rDNA transcriptional repression and nucleolar segregation, but the link between the two events remains unclear. Here we found that DSBs induced on rDNA trigger transcriptional repression in a cohesin- and HUSH (human silencing hub) complex-dependent manner throughout the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway. Altogether, our data indicate that rDNA break localization at the nucleolar periphery is not a direct consequence of transcriptional repression but rather is an active process that shares features with the mobilization of persistent DSB in active genes and heterochromatin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Ribossômico/genética , Regulação da Expressão Gênica/genética , RNA Longo não Codificante/metabolismo , Nucléolo Celular/metabolismo , Histonas/metabolismo , Recombinação Homóloga/genética , Membrana Nuclear/metabolismo , Coesinas
4.
Mol Cell ; 72(2): 250-262.e6, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30270107

RESUMO

Double-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.


Assuntos
Cromatina/genética , Reparo do DNA/genética , Histonas/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica/genética , Recombinação Homóloga/genética , Humanos , Células K562 , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
6.
Methods Mol Biol ; 2153: 427-438, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840796

RESUMO

Among the types of damage, DNA double-strand breaks (DSBs) (provoked by various environmental stresses, but also during normal cell metabolic activity) are the most deleterious, as illustrated by the variety of human diseases associated with DSB repair defects. DSBs are repaired by two groups of pathways: homologous recombination (HR) and nonhomologous end joining. These pathways do not trigger the same mutational signatures, and multiple factors, such as cell cycle stage, the complexity of the lesion and also the genomic location, contribute to the choice between these repair pathways. To study the usage of the HR machinery at DSBs, we propose a genome-wide method based on the chromatin immunoprecipitation of the HR core component Rad51, followed by high-throughput sequencing.


Assuntos
Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Sequenciamento Completo do Genoma/métodos , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Quebras de DNA de Cadeia Dupla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
7.
J Mol Biol ; 432(3): 724-736, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31401119

RESUMO

DNA double-strand breaks (DSBs) are harmful lesions that severely challenge genomic integrity, and recent evidence suggests that DSBs occur more frequently on the genome than previously thought. These lesions activate a complex and multilayered response called the DNA damage response, which allows to coordinate their repair with the cell cycle progression. While the mechanistic details of repair processes have been narrowed, thanks to several decades of intense studies, our knowledge of the impact of DSB on chromatin composition and chromosome architecture is still very sparse. However, the recent development of various tools to induce DSB at annotated loci, compatible with next-generation sequencing-based approaches, is opening a new framework to tackle these questions. Here we discuss the influence of initial and DSB-induced chromatin conformation and the strong potential of 3C-based technologies to decipher the contribution of chromosome architecture during DSB repair.


Assuntos
Cromatina/metabolismo , Cromatina/ultraestrutura , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/ultraestrutura , Conformação Molecular
8.
Nat Struct Mol Biol ; 24(4): 353-361, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28263325

RESUMO

The ability of DNA double-strand breaks (DSBs) to cluster in mammalian cells has been a subject of intense debate in recent years. Here we used a high-throughput chromosome conformation capture assay (capture Hi-C) to investigate clustering of DSBs induced at defined loci in the human genome. The results unambiguously demonstrated that DSBs cluster, but only when they are induced within transcriptionally active genes. Clustering of damaged genes occurs primarily during the G1 cell-cycle phase and coincides with delayed repair. Moreover, DSB clustering depends on the MRN complex as well as the Formin 2 (FMN2) nuclear actin organizer and the linker of nuclear and cytoplasmic skeleton (LINC) complex, thus suggesting that active mechanisms promote clustering. This work reveals that, when damaged, active genes, compared with the rest of the genome, exhibit a distinctive behavior, remaining largely unrepaired and clustered in G1, and being repaired via homologous recombination in postreplicative cells.


Assuntos
Mapeamento Cromossômico , Quebras de DNA de Cadeia Dupla , Genoma Humano , Linhagem Celular , Análise por Conglomerados , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , DNA Intergênico/genética , Fase G1/efeitos dos fármacos , Fase G1/genética , Histonas/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Domínios Proteicos , RNA Interferente Pequeno/metabolismo , Recombinação Genética/efeitos dos fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Transcrição Gênica/efeitos dos fármacos
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