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1.
Mol Cell ; 74(3): 571-583.e8, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30898438

RESUMO

In mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division.


Assuntos
Proteínas de Transporte/genética , Instabilidade Cromossômica/genética , Proteínas de Ligação a DNA/genética , Mitose/genética , Proteínas Nucleares/genética , Transativadores/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , Fase G1/genética , Genoma Humano/genética , Instabilidade Genômica/genética , Histonas , Humanos , Fosforilação , Transdução de Sinais/genética
2.
Mol Cell ; 73(3): 621-638.e17, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30554943

RESUMO

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Antineoplásicos/química , Azepinas/química , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Células K562 , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/química
3.
Trends Biochem Sci ; 45(4): 321-331, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32001093

RESUMO

DNA double-strand breaks (DSBs) are highly toxic lesions that can lead to chromosomal instability if they are not repaired correctly. DSBs are especially dangerous in mitosis when cells go through the complex process of equal chromosome segregation into daughter cells. When cells encounter DSBs in interphase, they are able to arrest the cell cycle until the breaks are repaired before entering mitosis. However, when DSBs occur during mitosis, cells no longer arrest but prioritize completion of cell division over repair of DNA damage. This review focuses on recent progress in our understanding of the mechanisms that allow mitotic cells to postpone DSB repair without accumulating massive chromosomal instability. Additionally, we review possible physiological consequences of failed DSB responses in mitosis.


Assuntos
Células/metabolismo , Mitose , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Humanos
4.
EMBO J ; 38(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30787182

RESUMO

DNA end resection initiates DNA double-strand break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step via MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In the absence of NBS1, MRE11-RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP augments MRE11: a phosphorylation-dependent mode through NBS1 and a phosphorylation-independent mode without NBS1. In support, we show that limited DNA end resection occurs in vivo in the absence of the FHA and BRCT domains of NBS1. Collectively, our data suggest that NBS1 restricts the MRE11-RAD50 nuclease to S-G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases , Recombinação Homóloga , Humanos , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/genética , Fosforilação
5.
Reprod Biol Endocrinol ; 17(1): 38, 2019 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-30981279

RESUMO

BACKGROUND: Matrix metalloproteinases (MMPs), especially the gelatinases MMP-2 and MMP-9, play a crucial role in the pathogenesis of endometriosis by enabling invasion. Doxycycline is a well-tolerated antibiotic and a potent MMP-inhibitor in subantimicrobial doses. METHODS: Gelatin zymography and activity assays were used to detect latent and active MMP-2 and -9 in cell culture supernatants of immortalized epithelial (12Z) and two isolates of primary endometriotic stromal cells treated with doxycycline. The invasiveness of 12Z endometriotic cells treated with doxycycline was assessed in matrigel-coated invasion chambers. The effect on latent and active MMP-2 expression of the combination of progesterone and doxycycline was tested in 12Z. RESULTS: Doxycycline significantly reduced the MMP-2 activity and pro-MMP-2 expression in 12Z and the MMP-2 and -9 activity as well as expression of pro-MMP-2 and -9 in primary endometriotic stromal cells. The percentage of 12Z cells invading through a matrigel-coated membrane was reduced to 65 and 22% of the control after treatment with doxycycline at doses of 1 µg/ml and 10 µg/ml, respectively. Furthermore, a combination of progesterone and doxycycline showed an additive effect in low doses on the reduction of MMP-2 activity and pro-MMP2 expression in 12Z endometriotic cells. CONCLUSIONS: In conclusion, the MMP-inhibiting features of subantimicrobial-dose doxycycline may be further evaluated as a well-tolerable additional therapeutic approach, e.g. in combination with progestins such as dienogest, in patients with infiltrative endometriosis with insufficient response to current medical treatment options.


Assuntos
Doxiciclina/farmacologia , Endometriose/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Células Estromais/efeitos dos fármacos
6.
Nucleic Acids Res ; 44(2): 538-44, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26615196

RESUMO

Maintenance of cellular homeostasis is key to prevent transformation and disease. The cellular response to DNA double-strand breaks, primarily orchestrated by the ATM/ATR kinases is one of many mechanisms that serve to uphold genome stability and homeostasis. Upon detection of double-strand breaks (DSBs), several signaling cascades are activated to halt cell cycle progression and initiate repair. Furthermore, the DNA damage response (DDR) controls cellular processes such as transcription, splicing and metabolism. Recent studies have uncovered aspects of how the DDR operates within nucleoli. It appears that the DDR controls transcription in the nucleoli, not only when DNA breaks occur in the rDNA repeats, but also when a nuclear DDR is activated. In addition, we have gained first insights into how repair of DSBs is organized in the nucleolus. Collectively, these recent studies provide a more comprehensive picture of how the DDR regulates basic cellular functions to maintain cellular homeostasis. In this review we will summarize recent findings and discuss their implications for our understanding of how the DDR regulates transcription and repair in the nucleolus.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Nucléolo Celular/genética , Reparo do DNA , DNA Ribossômico/genética , DNA/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Nucléolo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Ribossômico/metabolismo , Regulação da Expressão Gênica , Instabilidade Genômica , Homeostase/genética , Humanos , Transdução de Sinais , Transcrição Gênica
7.
BMC Biol ; 13: 30, 2015 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-25907681

RESUMO

BACKGROUND: Photoreceptor degeneration is a main hallmark of many blinding diseases making protection of photoreceptors crucial to prevent vision loss. Thus, regulation of endogenous neuroprotective factors may be key for cell survival and attenuation of disease progression. Important neuroprotective factors in the retina include H2O2 generated by injured photoreceptors, and leukemia inhibitory factor (LIF) expressed in Müller glia cells in response to photoreceptor damage. RESULTS: We present evidence that H2O2 connects to the LIF response by inducing stabilization of Lif transcripts in Müller cells. This process was independent of active gene transcription and p38 MAPK, but relied on AU-rich elements (AREs), which we identified within the highly conserved Lif 3'UTR. Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs. Among those, interleukin enhancer binding factor 3 (ILF3) was confirmed to participate in the redox-dependent Lif mRNA stabilization. Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells. CONCLUSIONS: Our results suggest that H2O2-induced redox signaling increases Lif transcript levels through ILF3 mediated mRNA stabilization. Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.


Assuntos
Células Ependimogliais/metabolismo , Fator Inibidor de Leucemia/metabolismo , Estabilidade de RNA/genética , Retina/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Camundongos , Oxirredução , Ratos , Degeneração Retiniana/metabolismo , Transdução de Sinais/genética
8.
Chromosoma ; 123(5): 423-36, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24861957

RESUMO

Senescence is a stable proliferation arrest characterized by profound changes in cellular morphology and metabolism as well as by extensive chromatin reorganization in the nucleus. One particular hallmark of chromatin changes during senescence is the formation of punctate DNA foci in DAPI-stained senescent cells that have been called senescence-associated heterochromatin foci (SAHF). While many advances have been made concerning our understanding of the effectors of senescence, how chromatin is reorganized and maintained in senescent cells has remained largely elusive. Because chromatin structure is inherently dynamic, senescent cells face the challenge of developing chromatin maintenance mechanisms in the absence of DNA replication in order to maintain the senescent phenotype. Here, we summarize and review recent findings shedding light on SAHF composition and formation via spatial repositioning of chromatin, with a specific focus on the role of lamin B1 for this process. In addition, we discuss the physiological implication of SAHF formation, the role of histone variants, and histone chaperones during senescence and also elaborate on the more general changes observed in the epigenome of the senescent cells.


Assuntos
Senescência Celular , Epigenômica , Heterocromatina/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Heterocromatina/genética , Histonas/genética , Histonas/metabolismo , Humanos
9.
Nucleic Acids Res ; 40(9): 3913-28, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22234878

RESUMO

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a 'head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/química , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Células Cultivadas , Proteínas Cromossômicas não Histona/análise , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/análise , Dimerização , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosfotreonina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Treonina/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
10.
Proc Natl Acad Sci U S A ; 108(36): 14944-9, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21896770

RESUMO

The bacterial pathogen Helicobacter pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The molecular mechanisms of H. pylori-associated gastric carcinogenesis remain ill defined. In this study, we examined the possibility that H. pylori directly compromises the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs) in primary and transformed murine and human epithelial and mesenchymal cells. The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA by pulse field gel electrophoresis and by high-magnification microscopy of metaphase chromosomes. Bacterial adhesion (e.g., via blood group antigen-binding adhesin) is required to induce DSBs; in contrast, the H. pylori virulence factors vacuolating cytotoxin A, γ-glutamyl transpeptidase, and the cytotoxin-associated gene (Cag) pathogenicity island are dispensable for DSB induction. The DNA discontinuities trigger a damage-signaling and repair response involving the sequential ataxia telangiectasia mutated (ATM)-dependent recruitment of repair factors--p53-binding protein (53BP1) and mediator of DNA damage checkpoint protein 1 (MDC1)--and histone H2A variant X (H2AX) phosphorylation. Although most breaks are repaired efficiently upon termination of the infection, we observe that prolonged active infection leads to saturation of cellular repair capabilities. In summary, we conclude that DNA damage followed by potentially imprecise repair is consistent with the carcinogenic properties of H. pylori and with its mutagenic properties in vitro and in vivo and may contribute to the genetic instability and frequent chromosomal aberrations that are a hallmark of gastric cancer.


Assuntos
Aderência Bacteriana , Quebras de DNA de Cadeia Dupla , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Aberrações Cromossômicas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Ilhas Genômicas , Infecções por Helicobacter/complicações , Infecções por Helicobacter/patologia , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Transativadores/genética , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
11.
J Clin Invest ; 133(23)2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37824211

RESUMO

An immunosuppressive microenvironment causes poor tumor T cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumors is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target, which when inhibited improved radiotherapy, even in tumors displaying immunosuppressive features and poor CD8+ T cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumor cell-specific functions. C5aR1 targeting resulted in increased NF-κB-dependent apoptosis specifically in tumors and not normal tissues, indicating that, in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumors and that targeting C5aR1 could improve radiotherapy, even in tumors displaying immunosuppressive features.


Assuntos
Complemento C5a , Receptores de Complemento , Humanos , Complemento C5a/genética , Receptores de Complemento/genética
12.
EMBO Rep ; 11(5): 387-92, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20224574

RESUMO

The MRE11-RAD50-NBS1 (MRN) complex accumulates at sites of DNA double-strand breaks in large chromatin domains flanking the lesion site. The mechanism of MRN accumulation involves direct binding of the Nijmegen breakage syndrome 1 (NBS1) subunit to phosphorylated mediator of the DNA damage checkpoint 1 (MDC1), a large nuclear adaptor protein that interacts directly with phosphorylated H2AX. NBS1 contains an FHA domain and two BRCT domains at its amino terminus. Here, we show that both of these domains participate in the interaction with phosphorylated MDC1. Point mutations in key amino acid residues of either the FHA or the BRCT domains compromise the interaction with MDC1 and lead to defects in MRN accumulation at sites of DNA damage. Surprisingly, only mutation in the FHA domain, but not in the BRCT domains, yields a G2/M checkpoint defect, indicating that MDC1-dependent chromatin accumulation of the MRN complex at sites of DNA breaks is not required for G2/M checkpoint activation.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Fase G2 , Humanos , Proteína Homóloga a MRE11 , Mitose , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Transativadores/química , Transativadores/metabolismo
13.
Nat Commun ; 13(1): 4143, 2022 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-35842428

RESUMO

The accurate repair of DNA double-strand breaks (DSBs), highly toxic DNA lesions, is crucial for genome integrity and is tightly regulated during the cell cycle. In mitosis, cells inactivate DSB repair in favor of a tethering mechanism that stabilizes broken chromosomes until they are repaired in the subsequent cell cycle phases. How this is achieved mechanistically is not yet understood, but the adaptor protein TOPBP1 is critically implicated in this process. Here, we identify CIP2A as a TOPBP1-interacting protein that regulates TOPBP1 localization specifically in mitosis. Cells lacking CIP2A display increased radio-sensitivity, micronuclei formation and chromosomal instability. CIP2A is actively exported from the cell nucleus in interphase but, upon nuclear envelope breakdown at the onset of mitosis, gains access to chromatin where it forms a complex with MDC1 and TOPBP1 to promote TOPBP1 recruitment to sites of mitotic DSBs. Collectively, our data uncover CIP2A-TOPBP1 as a mitosis-specific genome maintenance complex.


Assuntos
Autoantígenos , Proteínas de Transporte , Reparo do DNA , Proteínas de Ligação a DNA , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas Nucleares , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Instabilidade Cromossômica , DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitose/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
14.
Chromosoma ; 119(4): 337-49, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20224865

RESUMO

The chromatin structure is important for recognition and repair of DNA damage. Many DNA damage response proteins accumulate in large chromatin domains flanking sites of DNA double-strand breaks. The assembly of these structures-usually termed DNA damage foci-is primarily regulated by MDC1, a large nuclear mediator/adaptor protein that is composed of several distinct structural and functional domains. Here, we are summarizing the latest discoveries about the mechanisms by which MDC1 mediates DNA damage foci formation, and we are reviewing the considerable efforts taken to understand the functional implication of these structures.


Assuntos
Cromatina/metabolismo , Dano ao DNA , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Histonas/química , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos , Mitose , Proteínas Nucleares/química , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Transativadores/química
15.
DNA Repair (Amst) ; 108: 103215, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34455186

RESUMO

During mitosis, chromosomes undergo extensive structural changes resulting in the formation of compact cylindrical bodies and in the termination of the bulk of DNA-dependent metabolic activities. Therefore, DNA lesions that interfere with processes such as DNA replication and transcription in interphase are not expected to pose a major threat to genome stability in mitosis. There are, however, a few exceptions. DNA replication and repair intermediates that physically interconnect the sister chromatids jeopardize faithful chromosome segregation and need to be resolved before the onset of anaphase. In addition, dicentric chromosomes can form chromatin bridges and induce breakage-fusion-breakage cycles with dire consequences for genome stability. Finally, chromosome breaks that escape the G2/M DNA damage checkpoint or emerge early in mitosis may result in lagging acentric DNA fragments that mis-segregate and form micronuclei when cells exit from mitosis. Both chromatin bridges and micronuclei are potential sources of a mutational cascade that results in massive chromosomal instability and significantly contributes to genomic complexity. Here, we review recent progress in our understanding of the origins and consequences of chromosome bridges and micronuclei and the mechanisms by which cells suppress them.


Assuntos
Instabilidade Genômica , Mitose , Segregação de Cromossomos , Reparo do DNA , Replicação do DNA , Humanos
16.
Nat Cancer ; 2(12): 1357-1371, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-35121901

RESUMO

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.


Assuntos
Neoplasias , Mutações Sintéticas Letais , Proteínas de Transporte/genética , Instabilidade Cromossômica , DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , Recombinação Homóloga , Humanos , Proteínas Nucleares/genética
17.
DNA Repair (Amst) ; 8(7): 873-6, 2009 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-19446503

RESUMO

Histone H2A.X phosphorylation on Ser139 in response to DNA damage is the major signal for the assembly of the so-called gammaH2A.X chromatin domain, a region surrounding an unrepaired DNA double-strand break that is characterized by the accumulation of a large number of DNA damage response proteins. However, it is not yet clear how this event is regulated in space and time. The recent discovery of H2A.X Tyr142 phosphorylation by the WICH complex and its dephosphorylation by the EYA1/3 phosphatases may provide substantial novel insight into this process. WSTF, a subunit of the WICH complex bears a novel kinase domain at its N-terminus that constitutively targets H2A.X on Tyr142. This novel histone modification appears to determine the relative recruitment of either DNA repair or pro-apoptotic factors to sites of DNA damage. Thus, the balance of H2A.X Tyr142 phosphorylation/dephosphorylation may constitute a novel switch mechanism to determine cell fate after DNA damage.


Assuntos
Apoptose , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Tirosina/metabolismo , Animais , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Modelos Biológicos , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo
18.
iScience ; 23(10): 101594, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33205012

RESUMO

The importance of innate immunity in cancer is increasingly being recognized with recent reports suggesting tumor cell-intrinsic intracellular functions for innate immunity proteins. However, such functions are often poorly understood, and it is unclear whether these are affected by patient-specific mutations. Here, we show that C4b-binding protein alpha chain (C4BPA), typically thought to reside in the extracellular space, is expressed intracellularly in cancer cells, where it interacts with the NF-κB family member RelA and regulates apoptosis. Interestingly, intracellular C4BPA expression is regulated in a stress- and mutation-dependent manner and C4BPA mutations are associated with improved cancer survival outcome. Using cell lines harboring patient-specific C4BPA mutations, we show that increasing intracellular C4BPA levels correlate with sensitivity to oxaliplatin-induced apoptosis in vitro and in vivo. Mechanistically, sensitive C4BPA mutants display increased IκBα expression and increased inhibitory IκBα-RelA complex stability. These data suggest a non-canonical intracellular role for C4BPA in regulating NF-κB-dependent apoptosis.

19.
Nat Commun ; 11(1): 123, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913317

RESUMO

Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Nucléolo Celular/genética , DNA Ribossômico/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Motivos de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo
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