RESUMO
Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.
Assuntos
Cromatina/metabolismo , Quebras de DNA de Cadeia Simples , Reparo do DNA , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Cromatina/genética , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Especificidade por Substrato , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Xenopus laevisRESUMO
Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means of tandem Ub-binding domains, whereas it displays poor activity toward mono- or di-Ub substrates. In cells, ZUFSP is recruited to and regulates K63-Ub conjugates at genotoxic stress sites, promoting chromosome stability upon replication stress in a manner dependent on its catalytic activity. Our findings establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways.
Assuntos
Neoplasias Ósseas/enzimologia , Dano ao DNA , Enzimas Desubiquitinantes/metabolismo , Instabilidade Genômica , Osteossarcoma/enzimologia , Poliubiquitina/metabolismo , Epitélio Pigmentado da Retina/enzimologia , Sítios de Ligação , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/genética , Células HEK293 , Humanos , Lisina , Osteossarcoma/genética , Poliubiquitina/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato , UbiquitinaçãoRESUMO
DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)-mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error-free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error-free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR-mediated ICL repair is defective, and breaks are instead re-ligated by polymerase θ-dependent microhomology-mediated end-joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error-free ICL repair.
Assuntos
Anemia de Fanconi , DNA , Dano ao DNA , Reparo do DNA , Replicação do DNA , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , HumanosRESUMO
Dominant missense mutations in the human serine protease FAM111A underlie perinatally lethal gracile bone dysplasia and Kenny-Caffey syndrome, yet how FAM111A mutations lead to disease is not known. We show that FAM111A proteolytic activity suppresses DNA replication and transcription by displacing key effectors of these processes from chromatin, triggering rapid programmed cell death by Caspase-dependent apoptosis to potently undermine cell viability. Patient-associated point mutations in FAM111A exacerbate these phenotypes by hyperactivating its intrinsic protease activity. Moreover, FAM111A forms a complex with the uncharacterized homologous serine protease FAM111B, point mutations in which cause a hereditary fibrosing poikiloderma syndrome, and we demonstrate that disease-associated FAM111B mutants display amplified proteolytic activity and phenocopy the cellular impact of deregulated FAM111A catalytic activity. Thus, patient-associated FAM111A and FAM111B mutations may drive multisystem disorders via a common gain-of-function mechanism that relieves inhibitory constraints on their protease activities to powerfully undermine cellular fitness.
Assuntos
Doenças do Desenvolvimento Ósseo , Hiperostose Cortical Congênita , Proteínas de Ciclo Celular/genética , Mutação com Ganho de Função , Humanos , Mutação , Peptídeo Hidrolases , Receptores ViraisRESUMO
DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also known as UBE2N), an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains. Whereas RNF168 is known to catalyse ubiquitylation of H2A-type histones, leading to the recruitment of repair factors such as 53BP1 (refs 8-10), the critical substrates of RNF8 and K63-linked ubiquitylation remain elusive. Here we elucidate how RNF8 and UBC13 promote recruitment of RNF168 and downstream factors to DSB sites in human cells. We establish that UBC13-dependent K63-linked ubiquitylation at DSB sites is predominantly mediated by RNF8 but not RNF168, and that H1-type linker histones, but not core histones, represent major chromatin-associated targets of this modification. The RNF168 module (UDM1) recognizing RNF8-generated ubiquitylations is a high-affinity reader of K63-ubiquitylated H1, mechanistically explaining the essential roles of RNF8 and UBC13 in recruiting RNF168 to DSBs. Consistently, reduced expression or chromatin association of linker histones impair accumulation of K63-linked ubiquitin conjugates and repair factors at DSB-flanking chromatin. These results identify histone H1 as a key target of RNF8-UBC13 in DSB signalling and expand the concept of the histone code by showing that posttranslational modifications of linker histones can serve as important marks for recognition by factors involved in genome stability maintenance, and possibly beyond.
Assuntos
Dano ao DNA , Histonas/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Humanos , Lisina/metabolismo , Estrutura Terciária de Proteína , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: Tenting of the mitral leaflets is a major pathophysiological factor contributing to functional mitral regurgitation (FMR). A novel software tool allows automated quantification of the tenting volume (TnV) by 3D transesophageal echocardiography (TEE). The aims of this study are to investigate the correlations of biometric patient characteristics with the TnV and whether a threshold value for the diagnosis of a moderate or severe FMR can be calculated for the TnV. METHODS: This explorative and hypothesis-generating study analyzed the TnV of the mitral valve obtained by clinically indicated TEE. The mid-systolic, threefold calculated and averaged TnV from 80 patients with no or mild FMR and 27 patients with moderate or severe FMR was determined using the TomTec 4D MV Assessment tool. RESULTS: The TnV correlated significantly with the body size (r = 0.341), the weight (r = 0.272), and the body surface area (r = 0.320). After the adjustment to the body size, a threshold value of 1.25 cm3 /m was determined for the TnV by using a receiver-operating characteristic curve. This value distinguished moderate to severe from none to mild FMR with a sensitivity of 85% and a specificity of 71%. The intra-observer variability and inter-observer variability were determined to be 0.96 and 0.85, respectively. CONCLUSIONS: Automated assessment of TnV has the potential to support the diagnostic evaluation of FMR. Further studies are needed to validate this result, detect additional factors influencing the size of the TnV, and determine further thresholds for any degree of FMR.
Assuntos
Ecocardiografia Tridimensional , Insuficiência da Valva Mitral , Ecocardiografia Transesofagiana , Humanos , Valva Mitral/diagnóstico por imagem , Insuficiência da Valva Mitral/diagnóstico por imagem , Variações Dependentes do ObservadorRESUMO
Single-stranded DNA (ssDNA) regions form as an intermediate in many DNA-associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide-binding (OB) fold domain. The heterotrimeric, multi-OB fold domain-containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA-binding factor in human cells. RADX binds ssDNA via an N-terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA-binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA-binding proteins.
Assuntos
Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteína de Replicação A/genética , Sítios de Ligação , Linhagem Celular Tumoral , Dano ao DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Células HCT116 , Humanos , Modelos Moleculares , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína de Replicação A/química , Proteína de Replicação A/metabolismoRESUMO
BACKGROUND: Certain melanoma histotypes carry a worse prognosis than others. We aimed to identify patient related factors associated with specific melanoma histotypes. PATIENTS AND METHODS: Single center study including 347 melanoma patients, prospectively assessed for 22 variables leading to a database of more than 7,600 features. RESULTS: Melanomas were histologically categorized as superficial spreading (SSM, 70.6%), nodular (NM; 12.7%), acrolentiginous (ALM; 4.0%), lentigo maligna (LMM; 3.8%), or unclassified melanoma (UCM; 8.9%). Well recognized melanoma risk indicators (i. e. many atypical nevi, freckles, previous melanoma), were significantly associated with SSM and LMM histotypes. NM and ALM patients carried significantly less common and/or atypical nevi. NM were mostly self-detected or detected by relatives. In contrast, SSM, LMM, and ALM were most frequently detected by dermatologists. NM and UCM were preferentially located on poorly observable sites, SSM on the lower limbs, ALM on plantar sites, and LMM on the head and neck. ALM and LMM patients were significantly older than other patients. A multinomial logistic model was designed to predict a certain melanoma histotype (overall accuracy 81%), which could be helpful to focus the attention of clinicians or may be integrated into fully automated diagnostic algorithms. CONCLUSIONS: Melanoma histotypes show significant differences regarding patients' characteristics.
Assuntos
Dermoscopia/estatística & dados numéricos , Melanoma/epidemiologia , Melanoma/patologia , Modelos Estatísticos , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/patologia , Adulto , Distribuição por Idade , Idoso , Algoritmos , Simulação por Computador , Interpretação Estatística de Dados , Dermoscopia/métodos , Feminino , Alemanha/epidemiologia , Humanos , Masculino , Melanoma/classificação , Pessoa de Meia-Idade , Invasividade Neoplásica , Prevalência , Prognóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Medição de Risco/métodos , Sensibilidade e Especificidade , Distribuição por Sexo , Neoplasias Cutâneas/classificação , Adulto JovemRESUMO
Protein SUMOylation provides a principal driving force for cellular stress responses, including DNA-protein crosslink (DPC) repair and arsenic-induced PML body degradation. In this study, using genome-scale screens, we identified the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL), combining ubiquitin ligase activity through its RING domain with poly-SUMO binding via SUMO-interacting motifs, analogous to the STUbL RNF4. Mechanistically, TOPORS is a SUMO1-selective STUbL that complements RNF4 in generating complex ubiquitin landscapes on SUMOylated targets, including DPCs and PML, stimulating efficient p97/VCP unfoldase recruitment and proteasomal degradation. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, involving defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Our findings establish TOPORS as a STUbL whose parallel action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO-ubiquitin crosstalk.
RESUMO
The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discover that FAM104A, a protein of unknown function also named VCF1 (VCP/p97 nuclear Cofactor Family member 1), acts as a p97 cofactor in human cells. Detailed structure-function studies reveal that VCF1 directly binds p97 via a conserved α-helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.
Assuntos
Proteínas de Ciclo Celular , Ubiquitina , Humanos , Ligação Proteica , Ubiquitina/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Purpose: The purpose of this study was to determine a cutoff for progression of idiopathic full-thickness macular hole (MH) size. Methods: Retrospective analysis of consecutive patients waiting 4 weeks for MH surgery. Two observers performed 3 repeat sets of MH size measurements on optical coherence tomography (OCT) high-density radial scans taken at first presentation and 4 weeks later before surgery. Primary outcome was the definition of a cutoff for true enlargement of MH size versus measurement error. Secondary outcomes were risk factors for change in minimum linear diameter (MLD) size and best-corrected visual acuity (BCVA). Results: Fifty-one patients were included with a mean MH size of 334 µm (±179 µm; range 39 to 793 µm). The cutoff for an increase in MLD size calculated as the outer confidence limit for the 99.73% limits of agreement was 31 µm. This was independent of MH size. Using this cutoff, MLD size increased in 9/34 (26.5%) of patients without and in 14 of 17 (82.4%) of patients with vitreomacular traction (VMT; P < 0.001). Mean BCVA deteriorated in patients in whom the MH had progressed from 0.62 (±0.23) logMAR to 0.82 (±0.29; P < 0.001), whereas there was no significant change in BCVA in patients without MH progression (P = 0.25). In 31% (16/51) of patients, classification of their MHs (small ≤250 µm, medium 251-400 µm, and large >400 µm) changed over the 4-week period. Conclusions: Using a cutoff discriminates change from measurement error. A significant proportion of MHs progressed by 4 weeks, particularly in the presence of VMT. Translational Relevance: The established cutoff enables clinicians to differentiate true MH enlargement from measurement error.
Assuntos
Perfurações Retinianas , Humanos , Retina , Perfurações Retinianas/diagnóstico por imagem , Perfurações Retinianas/cirurgia , Estudos Retrospectivos , Tomografia de Coerência Óptica , Acuidade VisualRESUMO
The faithful segregation of eukaryotic chromosomes in mitosis requires that the genome be duplicated completely prior to anaphase. However, cells with large genomes sometimes fail to complete replication during interphase and instead enter mitosis with regions of incompletely replicated DNA. These regions are processed in early mitosis via a process known as mitotic DNA repair synthesis (MiDAS), but little is known about how cells switch from conventional DNA replication to MiDAS. Using the early embryo of the nematode Caenorhabditis elegans as a model system, we show that the TRAIP ubiquitin ligase drives replisome disassembly in response to incomplete DNA replication, thereby providing access to replication forks for other factors. Moreover, TRAIP is essential for MiDAS in human cells, and is important in both systems to prevent mitotic segregation errors. Our data indicate that TRAIP is a master regulator of the processing of incomplete DNA replication during mitosis in metazoa.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Reparo do DNA , Replicação do DNA , Mitose , Ubiquitina-Proteína Ligases/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Deleção de Genes , Humanos , Ubiquitina-Proteína Ligases/genéticaRESUMO
The ATR kinase is a master regulator of the cellular response to DNA replication stress. Activation of ATR relies on dual pathways involving the TopBP1 and ETAA1 proteins, both of which harbor ATR-activating domains (AADs). However, the exact contribution of the recently discovered ETAA1 pathway to ATR signaling in different contexts remains poorly understood. Here, using an unbiased CRISPR-Cas9-based genome-scale screen, we show that the ATR-stimulating function of ETAA1 becomes indispensable for cell fitness and chromosome stability when the fidelity of DNA replication is compromised. We demonstrate that the ATR-activating potential of ETAA1 is controlled by cell cycle- and replication stress-dependent phosphorylation of highly conserved residues within its AAD, and that the stimulatory impact of these modifications is required for the ability of ETAA1 to prevent mitotic chromosome abnormalities following replicative stress. Our findings suggest an important role of ETAA1 in protecting against genome instability arising from incompletely duplicated DNA via regulatory control of its ATR-stimulating potential.
Assuntos
Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Replicação do DNA , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Sistemas CRISPR-Cas , Ciclo Celular , Linhagem Celular Tumoral , Aberrações Cromossômicas , Dano ao DNA , Genoma Humano , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Mitose , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Transdução de SinaisRESUMO
When preparing young medical students for clinical activity, it is indispensable to acquaint them with anatomical section images which enable them to use the clinical application of imaging methods. A new Augmented Reality Magic Mirror (AR MM) system, which provides the advantage of a novel, interactive learning tool in addition to a regular dissection course, was therefore tested and evaluated by 880 first-year medical students as part of the macroscopic anatomy course in 2015/16 at Ludwig-Maximilians-Universität (LMU) in Munich. The system consists of an RGB-D sensor as a real-time tracking device, which enables the system to link a deposited section image to the projection of the user's body, as well as a large display mimicking a real-world physical mirror. Using gesture input, the users have the ability to interactively explore radiological images in different anatomical intersection planes. We designed a tutorial during which students worked with the system in groups of about 12 and evaluated the results. Subsequently, each participant was asked to assess the system's value by filling out a Likert-scale questionnaire. The respondents approved all statements which stressed the potential of the system to serve as an additional learning resource for anatomical education. In this case, emphasis was put on active learning, 3-dimensional understanding, and a better comprehension of the course of structures. We are convinced that such an AR MM system can be beneficially installed into anatomical education in order to prepare medical students more effectively for the clinical standards and for more interactive, student-centered learning.
Assuntos
Anatomia/educação , Estágio Clínico , Ensino , Educação de Graduação em Medicina , Humanos , Inquéritos e Questionários , Interface Usuário-ComputadorRESUMO
Maintenance of genome stability requires that DNA is replicated precisely once per cell cycle. This is believed to be achieved by limiting replication origin licensing and thereby restricting the firing of each replication origin to once per cell cycle. CDC6 is essential for eukaryotic replication origin licensing, however, it is poorly understood how CDC6 activity is constrained in higher eukaryotes. Here we report that the SCF(Cyclin F) ubiquitin ligase complex prevents DNA re-replication by targeting CDC6 for proteasomal degradation late in the cell cycle. We show that CDC6 and Cyclin F interact through defined sequence motifs that promote CDC6 ubiquitylation and degradation. Absence of Cyclin F or expression of a stable mutant of CDC6 promotes re-replication and genome instability in cells lacking the CDT1 inhibitor Geminin. Together, our work reveals a novel SCF(Cyclin F)-mediated mechanism required for precise once per cell cycle replication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclinas/metabolismo , Replicação do DNA , Proteínas Nucleares/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Motivos de Aminoácidos , Ciclo Celular , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Ciclinas/química , Ciclinas/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Proteólise , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/genéticaRESUMO
Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically, this requires a conserved domain in ETAA1 that potently and directly stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 gives rise to synthetic lethality characterized by massive genome instability and abrogation of ATR-dependent signalling. Our findings demonstrate that parallel TopBP1- and ETAA1-mediated pathways underlie ATR activation and that their combined action is essential for coping with replication stress.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , FosforilaçãoRESUMO
Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.
Assuntos
Replicação do DNA/genética , Instabilidade Genômica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Células Tumorais CultivadasRESUMO
Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Ciclinas/metabolismo , Reparo do DNA/fisiologia , Transativadores/metabolismo , Linhagem Celular Tumoral , Primers do DNA/genética , Citometria de Fluxo , Imunofluorescência , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Luciferases , Mutagênese Sítio-Dirigida , Interferência de RNA , RNA Interferente Pequeno/genética , UbiquitinaçãoRESUMO
The microtubule-dependent molecular motor KIF23 (Kinesin family member 23) is one of two components of the centralspindlin complex assembled during late stages of mitosis. Formation of this complex is known as an essential step for cytokinesis. Here, we identified KIF23 as a new transcriptional target gene of the tumor suppressor protein p53. We showed that p53 reduces expression of KIF23 on the mRNA as well as the protein level in different cell types. Promoter reporter assays revealed that this repression results from downregulation of KIF23 promoter activity. CDK inhibitor p21(WAF1/CIP1) was shown to be necessary to mediate p53-dependent repression. Furthermore, we identified the highly conserved cell cycle genes homology region (CHR) in the KIF23 promoter to be strictly required for p53-dependent repression as well as for cell cycle-dependent expression of KIF23. Cell cycle- and p53-dependent regulation of KIF23 appeared to be controlled by differential binding of DREAM and MMB complexes to the CHR element. With this study, we describe a new mechanism for transcriptional regulation of KIF23. Considering the strongly supporting function of KIF23 in cytokinesis, its p53-dependent repression may contribute to the prevention of uncontrolled cell growth.
Assuntos
Inativação Gênica , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Oncogênicas v-myb/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica , Células HCT116 , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Células NIH 3T3 , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta , Transcrição GênicaRESUMO
OBJECTIVES: To examine the feasibility and diagnostic value of a novel prehospital chest ultrasound algorithm in patients with dyspnea. METHODS: Sixty-two patients (32 men, 30 women, mean 67.1 years, range 20-90 years) with acute dyspnea prospectively underwent chest sonography with a portable ultrasound device. The algorithm included five sectional views (four-chamber subxyphoidal view, left and right laterodorsal view, left and right anterior intercostal space two to four view) screening for pleural and pericardial effusion, right heart distension, and pneumothorax. The prehospital sonographic findings were confirmed by chest radiograph, ultrasonography, and clinical follow-up in the emergency department. RESULTS: Prehospital chest emergency sonography trial was completed in 56 patients. Mean examination time was 2 min, and no scan took longer than 5 min. Sonography was easily integrated in the prehospital workflow alongside paramedic treatment without delay of treatment or transport. The most common diagnoses associated with acute dyspnea were (a) acute coronary syndrome (n=12, 21%), (b) decompensated congestive heart failure (CHF) (n=11, 20%), and (c) chronic obstructive pulmonary disease (COPD) (n=10, 18%). Pleural effusion was detected in 100% of CHF, 17% of acute coronary syndrome, and 20% of COPD patients, constituting a highly significant parameter in the differential diagnosis (P<0.01). Ultrasonography provided a helpful tool in n=38 (68%), and additional therapeutic consequences were drawn in n=14 (25%). CONCLUSION: Prehospital chest emergency sonography trial is a novel prehospital ultrasound algorithm for patients with dyspnea. Pleural effusion may serve as a novel prehospital marker for patients with decompensated CHF, thus facilitating the often difficult differential diagnosis between CHF and COPD.