RESUMO
Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A's recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end resection at DSBs, and its abrogation leads to upregulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning the complex DDR network for accurate and balanced execution of DSB repair.
Assuntos
Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Reparo de DNA por Recombinação/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Proteína BRCA1/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Células HeLa , Chaperonas de Histonas , Humanos , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
The nonhomologous end-joining (NHEJ) pathway is the primary repair pathway for DNA double strand breaks (DSBs) in humans. Repair is mediated by a core complex of NHEJ factors that includes a ligase (DNA Ligase IV; L4) that relies on juxtaposition of 3Î hydroxyl and 5Î phosphate termini of the strand breaks for catalysis. However, chromosome breaks arising from biological sources often have different end chemistries, and how these different end chemistries impact the way in which the core complex directs the necessary transitions from end pairing to ligation is not known. Here, using single-molecule FRET (smFRET), we show that prior to ligation, differences in end chemistry strongly modulate the bridging of broken ends by the NHEJ core complex. In particular, the 5Î phosphate group is a recognition element for L4 and is critical for the ability of NHEJ factors to promote stable pairing of ends. Moreover, other chemical incompatibilities, including products of aborted ligation, are sufficient to disrupt end pairing. Based on these observations, we propose a mechanism for iterative repair of DSBs by NHEJ.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/genética , DNA/metabolismo , Animais , DNA/química , Modelos Biológicos , Ligação ProteicaRESUMO
Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Western Blotting , Linhagem Celular Tumoral , DNA Ligase Dependente de ATP , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Humanos , CinéticaRESUMO
Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas F-Box/metabolismo , Recombinação Homóloga/fisiologia , Rad51 Recombinase/metabolismo , Animais , Células Cultivadas , Cromatina/enzimologia , Cromatina/genética , DNA/genética , DNA/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/citologia , Proteínas F-Box/genética , Humanos , Camundongos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Ligação Proteica , Rad51 Recombinase/genéticaRESUMO
PspA, -B and -C regulate the bacterial phage shock protein stress response by controlling the PspF transcription factor. Here, we have developed complementary approaches to study the behaviour of these proteins at their endogenous levels in Yersinia enterocolitica. First, we observed GFP-tagged versions with an approach that resolves individual protein complexes in live cells. This revealed that PspA, -B and -C share common behaviours, including a striking contrast before and after induction. In uninduced cells, PspA, -B and -C were highly mobile and widely distributed. However, induction reduced mobility and the proteins became more organized. Combining mCherry- and GFP-tagged proteins also revealed that PspA colocalizes with PspB and PspC into large stationary foci, often located close to the pole of induced cells. In addition, co-immunoprecipitation assays provided the first direct evidence supporting the model that PspA switches binding partners from PspF to PspBC upon induction. Together, these data suggest that PspA, -B and -C do not stably interact and are highly mobile before induction, perhaps sampling the status of the membrane and each other. However, an inducing signal promotes PspABC complex formation and their relocation to discrete parts of the membrane, which might then be important for mitigating envelope stress.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Estresse Fisiológico , Yersinia enterocolitica/fisiologia , Fusão Gênica Artificial , Membrana Celular/química , Genes Reporter , Imunoprecipitação , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão , Coloração e RotulagemRESUMO
Ventricular ATP-sensitive potassium (K(ATP)) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative K(ATP) channel-associated proteins. We investigated whether the association of K(ATP) channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co-immunoprecipitation experiments confirmed physical interaction between K(ATP) channels and PKP2 and plakoglobin in rat heart. Immunolocalization experiments demonstrated that K(ATP) channel subunits (Kir6.2 and SUR2A) are expressed at a higher density at the intercalated disk in mouse and rat hearts, where they co-localized with PKP2 and plakoglobin. Super-resolution microscopy demonstrate that K(ATP) channels are clustered within nanometer distances from junctional proteins. The local K(ATP) channel density, recorded in excised inside-out patches, was larger at the cell end when compared with local currents recorded from the cell center. The K(ATP) channel unitary conductance, block by MgATP and activation by MgADP, did not differ between these two locations. Whole cell K(ATP) channel current density (activated by metabolic inhibition) was â¼40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of K(ATP) channels was absent in the PKP2 deficient mice, but the K(ATP) channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of K(ATP) channel distribution within a cardiac myocyte. The higher K(ATP) channel density at the intercalated disk implies a possible role at the intercellular junctions during cardiac ischemia.
Assuntos
Canais KATP/química , Miócitos Cardíacos/citologia , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Animais , Membrana Celular/metabolismo , Desmossomos/metabolismo , Masculino , Camundongos , Microscopia/métodos , Isquemia Miocárdica/patologia , Placofilinas/metabolismo , Ratos , Ratos Sprague-Dawley , gama Catenina/metabolismoRESUMO
Sporadic Alzheimer's disease (AD) exclusively affects elderly people. Using direct conversion of AD patient fibroblasts into induced neurons (iNs), we generated an age-equivalent neuronal model. AD patient-derived iNs exhibit strong neuronal transcriptome signatures characterized by downregulation of mature neuronal properties and upregulation of immature and progenitor-like signaling pathways. Mapping iNs to longitudinal neuronal differentiation trajectory data demonstrated that AD iNs reflect a hypo-mature neuronal identity characterized by markers of stress, cell cycle, and de-differentiation. Epigenetic landscape profiling revealed an underlying aberrant neuronal state that shares similarities with malignant transformation and age-dependent epigenetic erosion. To probe for the involvement of aging, we generated rejuvenated iPSC-derived neurons that showed no significant disease-related transcriptome signatures, a feature that is consistent with epigenetic clock and brain ontogenesis mapping, which indicate that fibroblast-derived iNs more closely reflect old adult brain stages. Our findings identify AD-related neuronal changes as age-dependent cellular programs that impair neuronal identity.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Idoso , Envelhecimento , Fibroblastos , Humanos , NeurôniosRESUMO
Neurons are the longest-lived cells in our bodies and lack DNA replication, which makes them reliant on a limited repertoire of DNA repair mechanisms to maintain genome fidelity. These repair mechanisms decline with age, but we have limited knowledge of how genome instability emerges and what strategies neurons and other long-lived cells may have evolved to protect their genomes over the human life span. A targeted sequencing approach in human embryonic stem cell-induced neurons shows that, in neurons, DNA repair is enriched at well-defined hotspots that protect essential genes. These hotspots are enriched with histone H2A isoforms and RNA binding proteins and are associated with evolutionarily conserved elements of the human genome. These findings provide a basis for understanding genome integrity as it relates to aging and disease in the nervous system.
Assuntos
Reparo do DNA , Genoma Humano , Instabilidade Genômica , Neurônios/metabolismo , Envelhecimento/genética , Dano ao DNA , DNA Intergênico , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Células-Tronco Embrionárias , Histonas/metabolismo , Humanos , Mitose , Mutação , Doenças do Sistema Nervoso/genética , Neurônios/citologia , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de DNA , Transcrição GênicaRESUMO
One of the most central questions about the repair of a double-strand DNA break (DSB) concerns how the two free DNA ends are brought together - a step called synapsis. Using single-molecule FRET (smFRET), we show here that both Ku plus XRCC4:DNA ligase IV are necessary and sufficient to achieve a flexible synapsis of blunt DNA ends, whereas either alone is not. Addition of XLF causes a transition to a close synaptic state, and maximum efficiency of close synapsis is achieved within 20 min. The promotion of close synapsis by XLF indicates a role that is independent of a filament structure, with action focused at the very ends of each duplex. DNA-PKcs is not required for the formation of either the flexible or close synaptic states. This model explains in biochemical terms the evolutionarily central synaptic role of Ku, X4L4, and XLF in NHEJ for all eukaryotes.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Modelos Genéticos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de MoléculaRESUMO
Nonhomologous end joining (NHEJ) must adapt to diverse end structures during repair of chromosome breaks. Here, we investigate the mechanistic basis for this flexibility. DNA ends are aligned in a paired-end complex (PEC) by Ku, XLF, XRCC4, and DNA ligase IV (LIG4); we show by single-molecule analysis how terminal mispairs lead to mobilization of ends within PECs and consequent sampling of more end-alignment configurations. This remodeling is essential for direct ligation of damaged and mispaired ends during cellular NHEJ, since remodeling and ligation of such ends both require a LIG4-specific structural motif, insert1. Insert1 is also required for PEC remodeling that enables nucleolytic processing when end structures block direct ligation. Accordingly, cells expressing LIG4 lacking insert1 are sensitive to ionizing radiation. Cellular NHEJ of diverse ends thus identifies the steps necessary for repair through LIG4-mediated sensing of differences in end structure and consequent dynamic remodeling of aligned ends.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/metabolismo , Sequência de Aminoácidos , Reparo do DNA por Junção de Extremidades/efeitos da radiação , DNA Ligase Dependente de ATP/química , Humanos , Modelos Biológicos , Radiação IonizanteRESUMO
We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF for a single or a collection of images is estimated by first using stochastic modelling where the locations of clusters in the images are repeatedly randomized to estimate the distribution of the overlaps between the clusters in the absence of interaction (IF = 0). Second, an analytical form of the relationship between IF and the overlap (which has the random overlap as its only parameter) is used to estimate the IF for the experimentally observed overlap. The advantage of IF compared to conventional methods to quantify interaction in microscopy images is that it is insensitive to changing cluster density and is an absolute measure of interaction, making the interpretation of experiments easier. We validate the IF method by using both simulated and experimental data and provide an ImageJ plugin for determining the IF of an image.
Assuntos
Fenômenos Biofísicos , Microscopia/métodos , Processos Estocásticos , Análise por Conglomerados , Métodos , Imagem Molecular/métodosRESUMO
PARP1 is the main sensor of single- and double-strand breaks in DNA and, in building chains of poly(ADP-ribose), promotes the recruitment of many downstream signaling and effector proteins involved in the DNA damage response (DDR). We show a robust physical interaction between PARP1 and the replication fork protein TIMELESS, distinct from the known TIMELESS-TIPIN complex, which activates the intra-S phase checkpoint. TIMELESS recruitment to laser-induced sites of DNA damage is dependent on its binding to PARP1, but not PARP1 activity. We also find that the PARP1-TIMELESS complex contains a number of established PARP1 substrates, and TIMELESS mutants unable to bind PARP1 are impaired in their ability to bind PARP1 substrates. Further, PARP1 binding to certain substrates and their recruitment to DNA damage lesions is impaired by TIMELESS knockdown, and TIMELESS silencing significantly impairs DNA double-strand break repair. We hypothesize that TIMELESS cooperates in the PARP1-mediated DDR.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Ligação ProteicaRESUMO
AIMS: Cell function requires formation of molecular clusters localized to discrete subdomains. The composition of these interactomes, and their spatial organization, cannot be discerned by conventional microscopy given the resolution constraints imposed by the diffraction limit of light (â¼200-300 nm). Our aims were (i) Implement single-molecule imaging and analysis tools to resolve the nano-scale architecture of cardiac myocytes. (ii) Using these tools, to map two molecules classically defined as components 'of the desmosome' and 'of the gap junction', and defined their spatial organization. METHODS AND RESULTS: We built a set-up on a conventional inverted microscope using commercially available optics. Laser illumination, reducing, and oxygen scavenging conditions were used to manipulate the blinking behaviour of individual fluorescent reporters. Movies of blinking fluorophores were reconstructed to generate subdiffraction images at â¼20 nm resolution. With this method, we characterized clusters of connexin43 (Cx43) and of 'the desmosomal protein' plakophilin-2 (PKP2). In about half of Cx43 clusters, we observed overlay of Cx43 and PKP2 at the Cx43 plaque edge. SiRNA-mediated loss of Ankyrin-G expression yielded larger Cx43 clusters, of less regular shape, and larger Cx43-PKP2 subdomains. The Cx43-PKP2 subdomain was validated by a proximity ligation assay (PLA) and by Monte-Carlo simulations indicating an attraction between PKP2 and Cx43. CONCLUSIONS: (i) Super-resolution fluorescence microscopy, complemented with Monte-Carlo simulations and PLAs, allows the study of the nanoscale organization of an interactome in cardiomyocytes. (ii) PKP2 and Cx43 share a common hub that permits direct physical interaction. Its relevance to excitability, electrical coupling, and arrhythmogenic right ventricular cardiomyopathy, is discussed.
Assuntos
Conexina 43/análise , Desmossomos/química , Junções Comunicantes/química , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/ultraestrutura , Placofilinas/análise , Animais , Anquirinas/análise , Camundongos , Método de Monte Carlo , Miócitos Cardíacos/química , RatosRESUMO
In many human cancers, the tumor suppressor, p27(kip1) (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-ß-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer. The current studies show that treatment of endometrial carcinoma cells-1 (ECC-1) with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) stabilizes p27 in the nucleus, decreases p27 in the cytoplasm, and prevents E2-induced proliferation and degradation of p27 in endometrial carcinoma cells-1 and primary ECA cells. Furthermore, Skp2E3LIs increase p27 half-life by 6 hours, inhibit cell proliferation (IC50, 14.3µM), block retinoblastoma protein (pRB) phosphorylation, induce G1 phase block, and are not cytotoxic. Similarly, using super resolution fluorescence localization microscopy and quantification, Skp2E3LIs increase p27 protein in the nucleus by 1.8-fold. In vivo, injection of Skp2E3LIs significantly increases nuclear p27 and reduces proliferation of endometrial epithelial cells by 42%-62% in ovariectomized E2-primed mice. Skp2E3LIs are specific inhibitors of proteolytic degradation that pharmacologically target the binding interaction between the E3 ligase, SCF-Skp2/Cks1, and p27 to stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors have the potential to be an important therapeutic advance over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27.