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1.
Dev Cell ; 56(4): 461-477.e7, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33621493

RESUMO

Homology-directed repair (HDR) safeguards DNA integrity under various forms of stress, but how HDR protects replicating genomes under extensive metabolic alterations remains unclear. Here, we report that besides stalling replication forks, inhibition of ribonucleotide reductase (RNR) triggers metabolic imbalance manifested by the accumulation of increased reactive oxygen species (ROS) in cell nuclei. This leads to a redox-sensitive activation of the ATM kinase followed by phosphorylation of the MRE11 nuclease, which in HDR-deficient settings degrades stalled replication forks. Intriguingly, nascent DNA degradation by the ROS-ATM-MRE11 cascade is also triggered by hypoxia, which elevates signaling-competent ROS and attenuates functional HDR without arresting replication forks. Under these conditions, MRE11 degrades daughter-strand DNA gaps, which accumulate behind active replisomes and attract error-prone DNA polymerases to escalate mutation rates. Thus, HDR safeguards replicating genomes against metabolic assaults by restraining mutagenic repair at aberrantly processed nascent DNA. These findings have implications for cancer evolution and tumor therapy.


Assuntos
Replicação do DNA , Genoma Humano , Metabolismo , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA2/deficiência , Proteína BRCA2/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , DNA/metabolismo , Humanos , Proteína Homóloga a MRE11/metabolismo , Modelos Biológicos , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Polimerização , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
2.
Nature ; 587(7833): 297-302, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33087936

RESUMO

Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2-7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45-MCM-GINS (CMG) helicases that are required for genome duplication1-4. It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes5,6. Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3-7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue7,8. By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation.


Assuntos
Replicação do DNA/genética , Genoma Humano/genética , Proteínas de Manutenção de Minicromossomo/biossíntese , Proteínas de Manutenção de Minicromossomo/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Humanos , Proteínas de Manutenção de Minicromossomo/análise , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Estabilidade Proteica , Transporte Proteico
3.
Nature ; 574(7779): 571-574, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645724

RESUMO

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-POLα complex2. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Instabilidade Genômica , Conformação de Ácido Nucleico , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
4.
Nat Cell Biol ; 21(4): 487-497, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804506

RESUMO

Failure to complete DNA replication is a stochastic by-product of genome doubling in almost every cell cycle. During mitosis, under-replicated DNA (UR-DNA) is converted into DNA lesions, which are inherited by daughter cells and sequestered in 53BP1 nuclear bodies (53BP1-NBs). The fate of such cells remains unknown. Here, we show that the formation of 53BP1-NBs interrupts the chain of iterative damage intrinsically embedded in UR-DNA. Unlike clastogen-induced 53BP1 foci that are repaired throughout interphase, 53BP1-NBs restrain replication of the embedded genomic loci until late S phase, thus enabling the dedicated RAD52-mediated repair of UR-DNA lesions. The absence or malfunction of 53BP1-NBs causes premature replication of the affected loci, accompanied by genotoxic RAD51-mediated recombination. Thus, through adjusting replication timing and repair pathway choice at under-replicated loci, 53BP1-NBs enable the completion of genome duplication of inherited UR-DNA and prevent the conversion of stochastic under-replications into genome instability.


Assuntos
Estruturas do Núcleo Celular/fisiologia , Dano ao DNA , Período de Replicação do DNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia , Linhagem Celular , Segregação de Cromossomos , Reparo do DNA , Replicação do DNA , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética , Fase S/genética , Proteínas de Ligação a Telômeros/fisiologia
5.
Science ; 358(6364): 797-802, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29123070

RESUMO

DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)-generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathological consequences of replication stress. Thus, redox signaling couples fluctuations of dNTP biogenesis with replisome activity to reduce stress during genome duplication. We propose that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/genética , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ribonucleotídeo Redutases/metabolismo , Adaptação Biológica , Cromatina/metabolismo , Desoxirribonucleotídeos/metabolismo , Humanos , Redes e Vias Metabólicas , Oxirredução , Transdução de Sinais
6.
Nat Commun ; 7: 13887, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27976684

RESUMO

Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes.


Assuntos
Ciclo Celular , Proliferação de Células , Dano ao DNA/genética , Mitose/genética , Linhagem Celular Tumoral , Citocinese/genética , Quebras de DNA , Técnicas de Silenciamento de Genes , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Fenótipo , Imagem com Lapso de Tempo
7.
Nat Struct Mol Biol ; 23(8): 714-21, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27348077

RESUMO

Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair (HDR). Here, we show that silencing 53BP1 or exhausting its capacity to bind damaged chromatin changes limited DSB resection to hyper-resection and results in a switch from error-free gene conversion by RAD51 to mutagenic single-strand annealing by RAD52. Thus, rather than suppressing HDR, 53BP1 fosters its fidelity. These findings illuminate causes and consequences of synthetic viability acquired through 53BP1 silencing in cells lacking the BRCA1 tumor suppressor. We show that such cells survive DSB assaults at the cost of increasing reliance on RAD52-mediated HDR, which may fuel genome instability. However, our findings suggest that when challenged by DSBs, BRCA1- and 53BP1-deficient cells may become hypersensitive to, and be eliminated by, RAD52 inhibition.


Assuntos
Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Transporte Proteico , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo
8.
Nat Commun ; 6: 8088, 2015 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-26286827

RESUMO

Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation.


Assuntos
Poli Adenosina Difosfato Ribose/química , Proteínas/química , Proteínas/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Dano ao DNA , Regulação da Expressão Gênica/fisiologia , Humanos , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas/genética
9.
Nat Cell Biol ; 16(8): 792-803, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25064736

RESUMO

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Proteínas Nucleares/metabolismo , RNA Ribossômico/genética , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Nucléolo Celular/metabolismo , Sequência Conservada , Quebras de DNA de Cadeia Dupla , Inativação Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase I/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Genética
10.
Cell ; 155(5): 1088-103, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-24267891

RESUMO

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors.


Assuntos
Replicação do DNA , Instabilidade Genômica , Proteína de Replicação A/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Dano ao DNA/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Origem de Replicação
11.
Mol Cell ; 52(2): 206-20, 2013 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-24055346

RESUMO

Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling.


Assuntos
Cromatina/genética , Dano ao DNA , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas Associadas à Matriz Nuclear/genética , Receptores de Estrogênio/genética , Transdução de Sinais/genética , Acetilação , Western Blotting , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Microscopia de Fluorescência , Modelos Genéticos , Testes de Mutagenicidade , Proteínas Associadas à Matriz Nuclear/metabolismo , Fosforilação , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Interferência de RNA , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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