RESUMEN
It has been more than three decades since the discovery of multifunctional factors, the Non-POU-Domain-Containing Octamer-Binding Protein, NonO, and the Splicing Factor Proline- and Glutamine-Rich, SFPQ. Some of their functions, including their participation in transcriptional and posttranscriptional regulation as well as their contribution to paraspeckle subnuclear body organization, have been well documented. In this review, we focus on several other established roles of NonO and SFPQ, including their participation in the cell cycle, nonhomologous end-joining (NHEJ), homologous recombination (HR), telomere stability, childhood birth defects and cancer. In each of these contexts, the absence or malfunction of either or both NonO and SFPQ leads to either genome instability, tumor development or mental impairment.
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The ATR pathway plays a crucial role in maintaining genome integrity as the major DNA damage checkpoint. It also attracts attention as a therapeutic target in cancer treatment. The Rad17-RFC2-5 complex loads the Rad9-Hus1-Rad1 (9-1-1) DNA clamp complex onto damaged chromatin to activate the ATR pathway. We previously reported that phosphorylation of a polyanionic C-terminal tail of human Rad17, iVERGE, is essential for the interaction between Rad17 and the 9-1-1 complex. However, the molecular mechanism has remained unclear. Here, we show that iVERGE directly interacts with the Hus1 subunit of the 9-1-1 complex through Rad17-S667 phosphorylation independently of the AAA+ ATPase domains. An exogenous iVERGE peptide interacted with the 9-1-1 complex in vivo. The binding conformation of the iVERGE peptide was analyzed by de novo modeling with docking simulation, simulated annealing-molecular dynamics simulation, and the fragment molecular orbital method. The in silico analyses predicted the association of the iVERGE peptide with the hydrophobic and basic patches on the Hus1 protein, and the corresponding Hus1 mutants were deficient in the interaction with the iVERGE peptide in vivo. The iVERGE peptide occupied the same position as the C-terminus of Saccharomyces cerevisiae RAD24 on MEC3. The interaction energy calculation suggested that the Rad17 KYxxL motif and the iVERGE peptide are the primary and secondary interaction surfaces between the Rad17-RFC2-5 and 9-1-1 complexes. Our data reveal a novel molecular interface, iVERGE, between the Rad17-RFC2-5 and 9-1-1 complexes in vertebrates and implicate that Rad17 utilizes two distinct molecular interfaces to regulate the 9-1-1 complex.
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Adenosina Trifosfatasas , Cromatina , Humanos , Animales , Simulación de Dinámica Molecular , ATPasas Asociadas con Actividades Celulares Diversas , Proteínas de Ciclo CelularRESUMEN
The spindle position checkpoint (SPOC) is a mitotic surveillance mechanism in Saccharomyces cerevisiae that prevents cells from completing mitosis in response to spindle misalignment, thereby contributing to genomic integrity. The kinase Kin4, one of the most downstream SPOC components, is essential to stop the mitotic exit network (MEN), a signalling pathway that promotes the exit from mitosis and cell division. Previous work, however, suggested that a Kin4-independent pathway contributes to SPOC, yet the underlying mechanisms remain elusive. Here, we established the glycogen-synthase-kinase-3 (GSK-3) homologue Mck1, as a novel component that works independently of Kin4 to engage SPOC. Our data indicate that both Kin4 and Mck1 work in parallel to counteract MEN activation by the Cdc14 early anaphase release (FEAR) network. We show that Mck1's function in SPOC is mediated by the pre-replication complex protein and mitotic cyclin-dependent kinase (M-Cdk) inhibitor, Cdc6, which is degraded in a Mck1-dependent manner prior to mitosis. Moderate overproduction of Cdc6 phenocopies MCK1 deletion and causes SPOC deficiency via its N-terminal, M-Cdk inhibitory domain. Our data uncover an unprecedented role of GSK-3 kinases in coordinating spindle orientation with cell cycle progression.
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Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Humanos , Proteínas de Saccharomyces cerevisiae/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Huso Acromático/metabolismo , Saccharomycetales/metabolismo , Proteínas Serina-Treonina Quinasas , Fosforilación , Saccharomyces cerevisiae/genética , Mitosis , Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation.
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ARN Largo no Codificante , Fosforilación , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células Endoteliales/metabolismo , ADN/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismoRESUMEN
Topoisomerase II Binding Protein 1 (TOPBP1) is an important activator of the DNA damage response kinase Ataxia Telangiectasia and Rad3-related (ATR), although the mechanism by which this activation occurs is not yet known. TOPBP1 contains nine copies of the BRCA1 C-terminal repeat (BRCT) motif, which allows protein-protein and protein-DNA interactions. TOPBP1 also contains an ATR activation domain (AAD), which physically interacts with ATR and its partner ATR-interacting protein (ATRIP) in a manner that stimulates ATR kinase activity. It is unclear which of TOPBP1's nine BRCT domains participate in the reaction, as well as the individual roles played by these relevant BRCT domains. To address this knowledge gap, here, we delineated a minimal TOPBP1 that can activate ATR at DNA double-strand breaks in a regulated manner. We named this minimal TOPBP1 "Junior" and we show that Junior is composed of just three regions: BRCT0-2, the AAD, and BRCT7&8. We further defined the individual functions of these three regions by showing that BRCT0-2 is required for recruitment to DNA double-strand breaks and is dispensable thereafter, and that BRCT7&8 is dispensable for recruitment but essential to allow the AAD to multimerize and activate ATR. The delineation of TOPBP1 Junior creates a leaner, simplified, and better understood TOPBP1 and provides insight into the mechanism of ATR activation.
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Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Proteínas Nucleares , Proteínas de Xenopus , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Xenopus , Proteínas de Xenopus/metabolismoRESUMEN
BACKGROUND: Anti-PD1 checkpoint inhibitors (ICI) represent an established standard-of-care for patients with recurrent/metastatic head and neck squamous cell carcinoma (RMHNSCC). Landmark studies excluded patients with ECOG performance status (PS) ≥2; the benefit of ICI in this population is therefore unknown. METHODS: We retrospectively reviewed RMHNSCC patients who received 1+ dose of ICI at our institution between 2013 and 2019. Demographic and clinical data were obtained; the latter included objective response (ORR), toxicity, and any unplanned hospitalization (UH). Associations were explored using uni- and multivariate analysis. Overall survival (OS) was estimated using a Cox proportional hazards model; ORR, toxicity, and UH were evaluated with logistic regression. RESULTS: Of the 152 patients, 29 (19%) had an ECOG PS ≥2. Sixty-six (44%) experienced toxicity; 54 (36%) had a UH. A multivariate model for OS containing PS, smoking status, and HPV status demonstrated a strong association between ECOG ≥2 and shorter OS (p < 0.001; HR = 3.30, CI = 2.01-5.41). An association between OS and former (vs. never) smoking was also seen (p < 0.001; HR = 2.17, CI = 1.41-3.35); current smoking did not reach statistical significance. On univariate analysis, poor PS was associated with inferior ORR (p = 0.03; OR = 0.25, CI = 0.06-0.77) and increased UH (p = 0.04; OR = 2.43, CI = 1.05-5.71). There was no significant association between toxicity and any patient characteristic. CONCLUSIONS: We observed inferior OS, ORR, and rates of UH among ICI-treated RMHNSCC patients with ECOG 2/3. Our findings help frame discussion of therapeutic options in this poor-risk population.
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Carcinoma , Neoplasias de Cabeza y Cuello , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Estudios Retrospectivos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Carcinoma/tratamiento farmacológicoRESUMEN
Being sessile organisms, plants are ubiquitously exposed to stresses that can affect the DNA replication process or cause DNA damage. To cope with these problems, plants utilize DNA damage response (DDR) pathways, consisting of both highly conserved and plant-specific elements. As a part of this DDR, cell cycle checkpoint control mechanisms either pause the cell cycle, to allow DNA repair, or lead cells into differentiation or programmed cell death, to prevent the transmission of DNA errors in the organism through mitosis or to its offspring via meiosis. The two major DDR cell cycle checkpoints control either the replication process or the G2/M transition. The latter is largely overseen by the plant-specific SOG1 transcription factor, which drives the activity of cyclin-dependent kinase inhibitors and MYB3R proteins, which are rate limiting for the G2/M transition. By contrast, the replication checkpoint is controlled by different players, including the conserved kinase WEE1 and likely the transcriptional repressor RBR1. These checkpoint mechanisms are called upon during developmental processes, in retrograde signaling pathways, and in response to biotic and abiotic stresses, including metal toxicity, cold, salinity, and phosphate deficiency. Additionally, the recent expansion of research from Arabidopsis to other model plants has revealed species-specific aspects of the DDR. Overall, it is becoming evidently clear that the DNA damage checkpoint mechanisms represent an important aspect of the adaptation of plants to a changing environment, hence gaining more knowledge about this topic might be helpful to increase the resilience of plants to climate change.
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Absorción Fisiológica/genética , Arabidopsis/genética , Arabidopsis/fisiología , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/fisiología , Daño del ADN/genética , Estrés Fisiológico/genética , Absorción Fisiológica/fisiología , Daño del ADN/fisiología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Estrés Fisiológico/fisiología , Factores de TranscripciónRESUMEN
The deubiquitinating enzyme USP37 is known to contribute to timely onset of S phase and progression of mitosis. However, it is not clear if USP37 is required beyond S-phase entry despite expression and activity of USP37 peaking within S phase. We have utilized flow cytometry and microscopy to analyze populations of replicating cells labeled with thymidine analogs and monitored mitotic entry in synchronized cells to determine that USP37-depleted cells exhibited altered S-phase kinetics. Further analysis revealed that cells depleted of USP37 harbored increased levels of the replication stress and DNA damage markers γH2AX and 53BP1 in response to perturbed replication. Depletion of USP37 also reduced cellular proliferation and led to increased sensitivity to agents that induce replication stress. Underlying the increased sensitivity, we found that the checkpoint kinase 1 is destabilized in the absence of USP37, attenuating its function. We further demonstrated that USP37 deubiquitinates checkpoint kinase 1, promoting its stability. Together, our results establish that USP37 is required beyond S-phase entry to promote the efficiency and fidelity of replication. These data further define the role of USP37 in the regulation of cell proliferation and contribute to an evolving understanding of USP37 as a multifaceted regulator of genome stability.
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Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Endopeptidasas/metabolismo , Fase S , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Daño del ADN , Replicación del ADN , Endopeptidasas/genética , Estabilidad de Enzimas , Inestabilidad Genómica , Células HCT116 , Células HeLa , Histonas , Humanos , Células MCF-7 , UbiquitinaciónRESUMEN
The ATR pathway is one of the major DNA damage checkpoints, and Rad17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. Rad17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of Rad17 is important for recovery from the ATR pathway. Here, we identified several Rad17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of Rad17. Rad17 Δ230-270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of Rad17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of Rad17 is functionally linked to the proteasomal degradation. The ATP-binding activity of Rad17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The Rad17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of Rad17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of Rad17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of Rad17. We propose a model in which Rad17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage.
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Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Daño del ADN , Señales de Localización Nuclear/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Puntos de Control del Ciclo Celular , Células Cultivadas , Chlorocebus aethiops , Humanos , Señales de Localización Nuclear/química , Fosforilación , ProteolisisRESUMEN
Dna2 can efficiently process 5' flaps containing DNA secondary structure using coordinated action of the three biochemical activities: the N-terminally encoded DNA-binding activity and the C-terminally encoded endonuclease and helicase activities. In this study, we investigated the cross talk among the three functional domains using a variety of dna2 mutant alleles and enzymes derived thereof. We found that disruption of the catalytic activities of Dna2 activated Dna2-dependent checkpoint, residing in the N-terminal domain. This checkpoint activity contributed to growth defects of dna2 catalytic mutants, revealing the presence of an intramolecular functional cross talk in Dna2. The N-terminal domain of Dna2 bound specifically to substrates that mimic DNA replication fork intermediates, including Holliday junctions. Using site-directed mutagenesis of the N-terminal domain of Dna2, we discovered that five consecutive basic amino acid residues were essential for the ability of Dna2 to bind hairpin DNA in vitro. Mutant cells expressing the dna2 allele containing all five basic residues substituted with alanine displayed three distinct phenotypes: (i) temperature-sensitive growth defects, (ii) bypass of S-phase arrest, and (iii) increased sensitivity to DNA-damaging agents. Taken together, our results indicate that the interplay between the N-terminal regulatory and C-terminal catalytic domains of Dna2 plays an important role in vivo, especially when cells are placed under replication stress.
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ADN Helicasas/genética , Replicación del ADN/genética , ADN de Hongos/genética , Endonucleasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Biocatálisis , Ciclo Celular/genética , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN de Hongos/química , ADN de Hongos/metabolismo , Endonucleasas/química , Endonucleasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and kinetochores, much less is known regarding BuGZ's requirements. Here, we used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, we found that the kinetics of Bub1, BubR1, and BuGZ loading to kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to kinetochores, we carried out size-exclusion chromatography and analyzed Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions. We found that prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. Our results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling.
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Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Huso Acromático/metabolismo , Proteínas de Ciclo Celular/análisis , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/análisis , Proteínas de Unión a Poli-ADP-Ribosa/análisis , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Protein arginine methyltransferase 1 (PRMT1) is a key regulator of hepatic immune responses. Recently, we reported that PRMT1 regulates the tumor immune response in hepatocellular carcinoma (HCC). Here we found that PRMT1 expression in human HCC correlates with that of programmed cell death 1 ligand 1 (PD-L1), PD-L2, and other checkpoint genes. PRMT1 deletion in mice reduced PD-L1 and PD-L2 expression in tumors and reduced the efficiency of PD-1 antibody treatment in a diethylnitrosamine-induced HCC mouse model, suggesting that PRMT1 regulates the hepatic immune checkpoint. Mice had reduced PD-L1 and PD-L2 expression when PRMT1 was specifically deleted in tumor cells or macrophages, but PRMT1 deletion in dendritic cells did not alter PD-L1 and PD-L2 expression. rs975484 is a common polymorphism in the human PRMT1 gene promoter, and we found that it alters PRMT1 expression in blood monocytes and tumor-associated macrophages in human HCC. PRMT1 expression was higher in individuals with a GG genotype than in individuals with a CC genotype, and heterozygous carriers had intermediate expression. Luciferase reporter assays indicated that this differential expression is due to an extra C/EBPß-binding site in the PRMT1 promoter of individuals carrying the minor G allele. The rs975484 genotype also correlated with PRMT1 target expression in HCC. Individuals with the GG genotype had significantly higher levels of the PRMT1 targets PD-L1, PD-L2, and VISTA than those with the CC genotype. We conclude that PRMT1 critically controls immune checkpoints in mice and humans and that the PRMT1 polymorphism rs975484 affects checkpoint gene expression in HCC.
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Antígenos B7/inmunología , Antígeno B7-H1/inmunología , Carcinoma Hepatocelular/inmunología , Regulación Neoplásica de la Expresión Génica/inmunología , Neoplasias Hepáticas/inmunología , Proteínas de la Membrana/inmunología , Proteínas de Neoplasias/inmunología , Proteína 2 Ligando de Muerte Celular Programada 1/inmunología , Proteína-Arginina N-Metiltransferasas/inmunología , Proteínas Represoras/inmunología , Animales , Antígenos B7/genética , Antígeno B7-H1/genética , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Células Dendríticas/inmunología , Células Dendríticas/patología , Dietilnitrosamina/toxicidad , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Represoras/genética , Células THP-1RESUMEN
Galls induced by plant-parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such a background, potential DNA damage in the genome of gall cells is evident. We investigated whether DNA damage checkpoint activation followed by DNA repair occurred, or was eventually circumvented, in nematode-induced galls. Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1-knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1, were used to understand the mechanisms for coping with DNA damage in galls. Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely, disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted at the accumulation of mitotic defects. In addition, WEE1-knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root-knot nematodes. Together with data on DNA-damaging drugs, we suggest a conserved function for WEE1 in controlling G1/S cell cycle arrest in response to a replication defect in galls.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/parasitología , Ciclo Celular , Tumores de Planta/parasitología , Proteínas Serina-Treonina Quinasas/metabolismo , Tylenchoidea/fisiología , Animales , Arabidopsis/genética , Ciclo Celular/genética , Núcleo Celular/metabolismo , Daño del ADN , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Células Gigantes/citología , Glucuronidasa/metabolismo , Solanum lycopersicum/genética , Mitosis , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
DNA clamp, a highly conserved ring-shaped protein, binds dsDNA within its central pore. Also, DNA clamp interacts with various nuclear proteins on its front, thereby stimulating their enzymatic activities and biological functions. It has been assumed that the DNA clamp is a functionally single-faced ring from bacteria to humans. Here, we report the crystal structure of the heterotrimeric RAD9-RAD1-HUS1 (9-1-1) checkpoint clamp bound to a peptide of RHINO, a recently identified cancer-related protein that interacts with 9-1-1 and promotes activation of the DNA damage checkpoint. This is the first structure of 9-1-1 bound to its partner. The structure reveals that RHINO is unexpectedly bound to the edge and around the back of the 9-1-1 ring through specific interactions with the RAD1 subunit of 9-1-1. Our finding indicates that 9-1-1 is a functionally double-faced DNA clamp.
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Ciclo Celular , ADN/metabolismo , Péptidos/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Humanos , Modelos Moleculares , Péptidos/química , Unión ProteicaRESUMEN
Protein chains contain only l-amino acids, with the exception of the achiral glycine, making the chains homochiral. This homochirality is a prerequisite for proper protein folding and, hence, normal cellular function. The importance of d-amino acids as a component of the bacterial cell wall and their roles in neurotransmission in higher eukaryotes are well-established. However, the wider presence and the corresponding physiological roles of these specific amino acid stereoisomers have been appreciated only recently. Therefore, it is expected that enantiomeric fidelity has to be a key component of all of the steps in translation. Cells employ various molecular mechanisms for keeping d-amino acids away from the synthesis of nascent polypeptide chains. The major factors involved in this exclusion are aminoacyl-tRNA synthetases (aaRSs), elongation factor thermo-unstable (EF-Tu), the ribosome, and d-aminoacyl-tRNA deacylase (DTD). aaRS, EF-Tu, and the ribosome act as "chiral checkpoints" by preferentially binding to l-amino acids or l-aminoacyl-tRNAs, thereby excluding d-amino acids. Interestingly, DTD, which is conserved across all life forms, performs "chiral proofreading," as it removes d-amino acids erroneously added to tRNA. Here, we comprehensively review d-amino acids with respect to their occurrence and physiological roles, implications for chiral checkpoints required for translation fidelity, and potential use in synthetic biology.
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Proteínas Bacterianas/metabolismo , Biosíntesis de Proteínas/fisiología , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Bacterias/metabolismo , Pared Celular/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Ribosomas/metabolismo , EstereoisomerismoRESUMEN
Saccharomyces cerevisiae Tel1 is the ortholog of human ATM kinase and initiates a cell cycle checkpoint in response to dsDNA breaks (DSBs). Tel1ATM kinase is activated synergistically by naked dsDNA and the Mre11-Rad50-Xrs2NBS1 complex (MRX). A multisubunit protein complex, which is related to human shelterin, protects telomeres from being recognized as DSBs, thereby preventing a Tel1ATM checkpoint response. However, at very short telomeres, Tel1ATM can be recruited and activated by the MRX complex, resulting in telomere elongation. Conversely, at long telomeres, Rap1-interacting-factor 2 (Rif2) is instrumental in suppressing Tel1 activity. Here, using an in vitro reconstituted Tel1 kinase activation assay, we show that Rif2 inhibits MRX-dependent Tel1 kinase activity. Rif2 discharges the ATP-bound form of Rad50, which is essential for all MRX-dependent activities. This conclusion is further strengthened by experiments with a Rad50 allosteric ATPase mutant that maps outside the conserved ATP binding pocket. We propose a model in which Rif2 attenuates Tel1 activity at telomeres by acting directly on Rad50 and discharging its activated ATP-bound state, thereby rendering the MRX complex incompetent for Tel1 activation. These findings expand our understanding of the mechanism by which Rif2 controls telomere length.
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Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismoRESUMEN
Sequential activation of DNA replication origins is precisely programmed and critical to maintaining genome stability. RecQL4, a member of the conserved RecQ family of helicases, plays an essential role in the initiation of DNA replication in mammalian cells. Here, we showed that RecQL4 protein tethered on the pre-replicative complex (pre-RC) induces early activation of late replicating origins during S phase. Tethering of RecQL4 or its N terminus on pre-RCs via fusion with Orc4 protein resulted in the recruitment of essential initiation factors, such as Mcm10, And-1, Cdc45, and GINS, increasing nascent DNA synthesis in late replicating origins during early S phase. In this origin activation process, tethered RecQL4 was able to recruit Cdc45 even in the absence of cyclin-dependent kinase (CDK) activity, whereas CDK phosphorylation of RecQL4 N terminus was required for interaction with and origin recruitment of And-1 and GINS. In addition, forced activation of replication origins by RecQL4 tethering resulted in increased replication stress and the accumulation of ssDNAs, which can be recovered by transcription inhibition. Collectively, these results suggest that recruitment of RecQL4 to replication origins is an important step for temporal activation of replication origins during S phase. Further, perturbation of replication timing control by unscheduled origin activation significantly induces replication stress, which is mostly caused by transcription-replication conflicts.
Asunto(s)
Replicación del ADN , RecQ Helicasas/metabolismo , Origen de Réplica , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Complejo de Reconocimiento del Origen/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , RecQ Helicasas/genética , Fase S , Activación TranscripcionalRESUMEN
The response of cancer cells to therapeutic drugs that cause DNA damage depends on genes playing a role in DNA repair. RecQ-like helicase 1 (RECQ1), a DNA repair helicase, is critical for genome stability, and loss-of-function mutations in the RECQ1 gene are associated with increased susceptibility to breast cancer. In this study, using a CRISPR/Cas9-edited cell-based model, we show that the genetic or functional loss of RECQ1 sensitizes MDA-MB-231 breast cancer cells to gemcitabine, a nucleoside analog used in chemotherapy for triple-negative breast cancer. RECQ1 loss led to defective ATR Ser/Thr kinase (ATR)/checkpoint kinase 1 (ChK1) activation and greater DNA damage accumulation in response to gemcitabine treatment. Dual deficiency of MUS81 structure-specific endonuclease subunit (MUS81) and RECQ1 increased gemcitabine-induced, replication-associated DNA double-stranded breaks. Consistent with defective checkpoint activation, a ChK1 inhibitor further sensitized RECQ1-deficient cells to gemcitabine and increased cell death. Our results reveal an important role for RECQ1 in controlling cell cycle checkpoint activation in response to gemcitabine-induced replication stress.
Asunto(s)
Antineoplásicos/toxicidad , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Desoxicitidina/análogos & derivados , RecQ Helicasas/metabolismo , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxicitidina/toxicidad , Endonucleasas/genética , Endonucleasas/metabolismo , Humanos , RecQ Helicasas/genética , GemcitabinaRESUMEN
In Saccharomyces cerevisiae, Tel1 protein kinase, the ortholog of human ataxia telangiectasia-mutated (ATM), is activated in response to DNA double-strand breaks. Biochemical studies with human ATM and genetic studies in yeast suggest that recruitment and activation of Tel1ATM depends on the heterotrimeric MRXMRN complex, composed of Mre11, Rad50, and Xrs2 (human Nbs1). However, the mechanism of activation of Tel1 by MRX remains unclear, as does the role of effector DNA. Here we demonstrate that dsDNA and MRX activate Tel1 synergistically. Although minimal activation was observed with 80-mer duplex DNA, the optimal effector for Tel1 activation is long, nucleosome-free DNA. However, there is no requirement for DNA double-stranded termini. The ATPase activity of Rad50 is critical for activation. In addition to DNA and Rad50, either Mre11 or Xrs2, but not both, is also required. Each of the three MRX subunits shows a physical association with Tel1. Our study provides a model of how the individual subunits of MRX and DNA regulate Tel1 kinase activity.
Asunto(s)
ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Nucleosomas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Roturas del ADN de Doble Cadena , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In all eukaryotes, a functional mitotic spindle is essential for distributing duplicated chromosomes into daughter cells. Mitotic spindle assembly involves highly ordered arrangement of microtubules (MTs). The Augmin protein complex recruits γ-tubulin ring complex (γ-TuRC) to MTs and thereby promotes MT-based MT nucleation and mitotic spindle assembly. However, several factors that may promote Augmin recruitment to MTs remain unknown. Here, we show that echinoderm microtubule-associated protein-like 3 (EML3), an MT-associated protein, facilitates binding between MTs and Augmin/γ-TuRC and recruiting the latter to MTs for proper mitotic spindle assembly and kinetochore-MT connections. Using immunofluorescence microscopy, live-cell imaging, and immunoprecipitation assays, we found that EML3 recruits Augmin/γ-TuRC to the MTs to enhance MT-based MT nucleation in both spindle and small acentrosomal asters. We also noted that the EML3-mediated recruitment is controlled by cyclin-dependent kinase 1 (CDK1), which phosphorylated EML3 at Thr-881 and promoted its binding to Augmin/γ-TuRC. RNAi-mediated EML3 knockdown in HeLa cells reduced spindle localization of Augmin/γ-TuRC, which resulted in abnormal spindle assembly and caused kinetochore-MT misconnection. The introduction of exogenous WT or a Thr-881 phosphorylation mimic EML3 variant into the EML3 knockdown cells restored normal Augmin/γ-TuRC localization and spindle assembly. The EML3 knockdown also affected the spindle assembly checkpoint, delaying chromosome congression and cell division. Taken together, our results indicate that EML3 regulates mitotic spindle assembly and the kinetochore-MT connection by regulating MT-based MT nucleation and recruiting Augmin/γ-TuRC to MTs.