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Background: Current radiotherapy regimens for glioblastoma (GBM) have limited efficacy and fails to eradicate tumors. Regenerative medicine brings hope for repairing damaged tissue, opening opportunities for elevating the maximum acceptable radiation dose. In this study, we explored the effect of ultra-high dose fractionated radiation on tumor responses and brain injury in immunocompetent mice which can better mimic the tumor-host interactions observed in patients. We also evaluated the role of the hypoxia-inducible factor-1 alpha under radiation as potential target for combating radiation-induced brain injury. Methods: Naïve and Hif-1α+/- heterozygous mice received a fractionated daily dose of 20 Gy for three or five consecutive days. Magnetic resonance imaging (MRI) and histology were performed to assess brain injury post-radiation. The 2×105 human GBM1 luciferase-expressing cells were transplanted with tolerance induction protocol. Fractionated radiotherapy was performed during the exponential phase of tumor growth. Bioluminescence imaging, MRI, and immunohistochemistry staining were performed to evaluate tumor growth dynamics and radiotherapy responses. Additionally, animal lifespan was recorded. Results: Fractionated radiation of 5×20 Gy induced severe brain damage, starting 3 weeks after radiation. All animals from this group died within 12 weeks. In contrast, later onset and less severe brain injury were observed starting 12 weeks after radiation of 3×20 Gy. It resulted in complete GBM eradication and survival of all treated animals. Furthermore, Hif-1α+/- mice exhibited more severe vascular damage after fractionated radiation of 3×20 Gy. Conclusion: Ultra-high dose fractionated 3×20 Gy radiation has the potential to fully eradicate GBM cells at the cost of only mild brain injury. The Hif-1α gene is a promising target for ameliorating vascular impairment post-radiation, encouraging the implementation of neurorestorative strategies.
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Telomeric repeat-containing RNA (TERRA) and its formation of RNA:DNA hybrids (or TERRA R-loops), influence telomere maintenance, particularly in human cancer cells that use homologous recombination-mediated alternative lengthening of telomeres. Here, we report that the RNA-binding motif protein 14 (RBM14) is associated with telomeres in human cancer cells. RBM14 negatively regulates TERRA expression. It also binds to TERRA and inhibits it from forming TERRA R-loops at telomeres. RBM14 depletion has several effects, including elevated TERRA levels, telomeric R-loops, telomere dysfunction-induced DNA damage foci formation, particularly in the presence of DNA replication stress, pRPA32 accumulation at telomeres and telomere signal-free ends. Thus, RBM14 protects telomere integrity via modulating TERRA levels and its R-loop formation at telomeres.
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Homeostasis del Telómero , Telómero , Humanos , ADN/genética , Estructuras R-Loop , ARN/genética , ARN/metabolismo , ARN Largo no Codificante/genética , Motivos de Unión al ARN , Telómero/genética , Telómero/metabolismo , Neoplasias/genéticaRESUMEN
Background: Conventional radiation therapy for glioblastoma (GBM) has limited efficacy. Regenerative medicine brings hope for repairing damaged tissue, opening opportunities for elevating the maximum acceptable radiation dose. In this study, we explored the effect of ultra-high dose fractionated radiation on brain injury and tumor responses in immunocompetent mice. We also evaluated the role of the HIF-1α under radiation. Methods: Naïve and hypoxia-inducible factor-1 alpha (HIF-1α)+/- heterozygous mice received a fractionated daily dose of 20 Gy for three or five consecutive days. Magnetic resonance imaging (MRI) and histology were performed to assess brain injury post-radiation. The 2×105 human GBM1 luciferase-expressing cells were transplanted with tolerance induction protocol. Fractionated radiotherapy was performed during the exponential phase of tumor growth. BLI, MRI, and immunohistochemistry staining were performed to evaluate tumor growth dynamics and radiotherapy responses. Additionally, animal lifespan was recorded. Results: Fractionated radiation of 5×20 Gy induced severe brain damage, starting 3 weeks after radiation. All animals from this group died within 12 weeks. In contrast, later onset and less severe brain injury were observed starting 12 weeks after radiation of 3×20 Gy. It resulted in complete GBM eradication and survival of all treated animals. Furthermore, HIF-1α+/- mice exhibited more obvious vascular damage 63 weeks after fractionated radiation of 3×20 Gy. Conclusion: Ultra-high dose fractionated 3×20 Gy radiation can eradicate the GBM cells at the cost of only mild brain injury. The HIF-1α gene is a promising target for ameliorating vascular impairment post-radiation, encouraging the implementation of neurorestorative strategies.
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Currently, human glioma tumors are mostly modeled in immunodeficient recipients; however, lack of interactions with adaptive immune system is a serious flaw, particularly in the era when immunotherapies dominate treatment strategies. Our group was the first to successfully establish the orthotopic transplantation of human glioblastoma (GBM) in immunocompetent mice by inducing immunological tolerance using a short-term, systemic costimulation blockade strategy (CTLA-4-Ig and MR1). In this study, we further validated the feasibility of this method by modeling pediatric diffuse intrinsic pontine glioma (DIPG) and two types of adult GBM (GBM1, GBM551), in mice with intact immune systems and immunodeficient mice. We found that all three glioma models were successfully established, with distinct difference in tumor growth patterns and morphologies, after orthotopic xenotransplantation in tolerance-induced immunocompetent mice. Long-lasting tolerance that is maintained for up to nearly 200 d in GBM551 confirmed the robustness of this model. Moreover, we found that tumors in immunocompetent mice displayed features more similar to the clinical pathophysiology found in glioma patients, characterized by inflammatory infiltration and strong neovascularization, as compared with tumors in immunodeficient mice. In summary, we have validated the robustness of the costimulatory blockade strategy for tumor modeling and successfully established three human glioma models including the pediatric DIPG whose preclinical study is particularly thwarted by the lack of proper animal models.
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Neoplasias del Tronco Encefálico , Glioblastoma , Glioma , Adulto , Animales , Niño , Humanos , Ratones , Neovascularización PatológicaRESUMEN
PURPOSE: Improved efficacy of anticancer therapy and a growing pool of survivors give rise to a question about their quality of life and return to premorbid status. Radiation is effective in brain metastasis eradication, although the optimal approach and long-term effects on brain function are largely unknown. We studied the effects of radiosurgery on brain function. METHODS AND MATERIALS: Adult C57BL/6J mice with or without brain metastases (rat 9L gliosarcoma) were treated with cone beam single-arc stereotactic radiosurgery (SRS; 40 Gy). Tumor growth was monitored using bioluminescence, whereas longitudinal magnetic resonance imaging, behavioral studies, and histologic analysis were performed to evaluate brain response to the treatment for up to 18 months. RESULTS: Stereotactic radiosurgery (SRS) resulted in 9L metastases eradication within 4 weeks with subsequent long-term survival of all treated animals, whereas all nontreated animals succumbed to the brain tumor. Behavioral impairment, as measured with a recognition memory test, was observed earlier in mice subjected to radiosurgery of tumors (6 weeks) in comparison to SRS of healthy brain tissue (10 weeks). Notably, the deficit resolved by 18 weeks only in mice not bearing a tumor, whereas tumor eradication was complicated by the persistent cognitive deficits. In addition, the results of magnetic resonance imaging were unremarkable in both groups, and histopathology revealed changes. SRS-induced tumor eradication triggered long-lasting and exacerbated neuroinflammatory response. No demyelination, neuronal loss, or hemorrhage was detected in any of the groups. CONCLUSIONS: Tumor disintegration by SRS leads to exacerbated neuroinflammation and persistent cognitive deficits; therefore, methods aiming at reducing inflammation after tumor eradication or other therapeutic methods should be sought.
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Neoplasias Encefálicas/radioterapia , Encéfalo/efectos de la radiación , Disfunción Cognitiva/etiología , Gliosarcoma/radioterapia , Radiocirugia/efectos adversos , Animales , Atención/efectos de la radiación , Conducta , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Disfunción Cognitiva/diagnóstico por imagen , Encefalitis/diagnóstico por imagen , Encefalitis/etiología , Encefalitis/patología , Gliosarcoma/mortalidad , Gliosarcoma/patología , Gliosarcoma/secundario , Gliosis/etiología , Mediciones Luminiscentes , Activación de Macrófagos , Imagen por Resonancia Magnética/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias/métodos , Radiocirugia/métodos , Dosificación Radioterapéutica , Reconocimiento en PsicologíaRESUMEN
Accumulating evidence suggests participation of RNA-binding proteins with intrinsically disordered domains (IDPs) in the DNA damage response (DDR). These IDPs form liquid compartments at DNA damage sites in a poly(ADP ribose) (PAR)-dependent manner. However, it is greatly unknown how the IDPs are involved in DDR. We have shown previously that one of the IDPs RBM14 is required for the canonical nonhomologous end joining (cNHEJ). Here we show that RBM14 is recruited to DNA damage sites in a PARP- and RNA polymerase II (RNAPII)-dependent manner. Both KU and RBM14 are required for RNAPII-dependent generation of RNA:DNA hybrids at DNA damage sites. In fact, RBM14 binds to RNA:DNA hybrids. Furthermore, RNA:DNA hybrids and RNAPII are detected at gene-coding as well as at intergenic areas when double-strand breaks (DSBs) are induced. We propose that the cNHEJ pathway utilizes damage-induced transcription and intrinsically disordered protein RBM14 for efficient repair of DSBs.
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Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Quimera , Células HEK293 , Humanos , Autoantígeno Ku/metabolismo , Hibridación de Ácido Nucleico , Dominios Proteicos , ARN/genética , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
DNA methylation has long been recognized as a tumor-promoting factor when aberrantly regulated in the promoter region of genes. However, the effect of intragenic DNA methylation remains poorly understood on the clinical aspects of cancer. Here, we first evaluated the significance of intragenic DNA methylation for survival outcomes of cancer patients in a genome-wide manner. Glioblastoma patients with hypermethylated intragenic regions exhibited better survival than hypomethylated patients. Enrichment analyses of intragenic DNA methylation profiles with epigenetic signatures prioritized the intragenic DNA methylation of ZMIZ1 as a possible glioblastoma prognostic marker that is independent of MGMT methylation in IDH1 wild-type patients. This intragenic region harbored molecular signatures of alternative transcription across many cell types. Furthermore, we found that the intragenic region of ZMIZ1 can serve as a molecular marker in multiple cancers including astrocytomas, bladder cancer and renal cell carcinoma according to DNA methylation status. Finally, in vitro and in vivo experiments uncovered the role of ZMIZ1 as a driver of tumor cell migration. Altogether, our results identify ZMIZ1 as a prognostic marker in cancer and highlight the clinical significance of intragenic methylation in cancer.
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Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Metilación de ADN/genética , Glioblastoma/genética , Glioblastoma/patología , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Epigénesis Genética/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo/métodos , Ratones Desnudos , Pronóstico , Regiones Promotoras Genéticas/genética , Transcripción Genética/genéticaRESUMEN
Defects in the DNA damage response (DDR) are associated with multiple diseases, including cancers and neurodegenerative disorders. Emerging evidence indicates involvement of RNA-binding proteins (RBPs) in DDR. However, functions of RBPs in the DDR pathway remain elusive. We have shown previously that the RNA-binding protein RBM14 is required for non-homologous end joining (NHEJ). Here we show that RBM14 is required for efficient recruitment of XRCC4 and XLF to chromatin and the release of KU proteins from chromatin upon DNA damage. Failure of this process leads to accumulation of double-strand breaks (DSBs) in cells. Thus RBM14 plays crucial role in regulation of NHEJ upon DNA damage.
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Péptidos y Proteínas de Señalización Intracelular/fisiología , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Fosforilación , Unión Proteica , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Transporte de ProteínasRESUMEN
The author would like to insert the citation after the following sentence, "These RBPs are detected on laser trackswithin one minute after laser irradiation, and are excluded from the laser tracks shortly (within 10-15 min, depending on conditions of laser irradiation) [1]", in the paper published in the International Journal of Molecular Sciences [2].[...].
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Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, "sensor" proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)'s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as "liquid-demixing"). Among the PAR-associated IDPs are FUS/TLS (fused in sarcoma/translocated in sarcoma), EWS (Ewing sarcoma), TARF15 (TATA box-binding protein-associated factor 68 kDa) (also called FET proteins), a number of heterogeneous nuclear ribonucleoproteins (hnRNPs), and RBM14. Importantly, various point mutations within the FET genes have been implicated in pathological protein aggregation in neurodegenerative diseases, specifically with amyotrophic lateral sclerosis (ALS), and frontotemporal lobe degeneration (FTLD). The FET proteins also frequently exhibit gene translocation in human cancers, and emerging evidence shows their physical interactions with DDR proteins and thus implies their involvement in the maintenance of genome stability.
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Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Animales , Proteínas de Unión al ADN/metabolismo , Humanos , Transducción de SeñalRESUMEN
DNA damage activates the cell cycle checkpoint to regulate cell cycle progression. The checkpoint clamp (Rad9-Hus1-Rad1 complex) is recruited to damage sites, and is required for checkpoint activation. While functions of the checkpoint clamp in checkpoint activation have been well studied, its functions in DNA repair regulation remain elusive. Here we show that Rad9 is required for efficient homologous recombination (HR), and facilitates DNA-end resection. The role of Rad9 in homologous recombination is independent of its function in checkpoint activation, and this function is important for preventing alternative non-homologous end joining (altNHEJ). These findings reveal novel function of the checkpoint clamp in HR.
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Proteínas de Ciclo Celular/metabolismo , Reparación del ADN por Unión de Extremidades , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN , ADN Helicasas/metabolismo , Recombinación Homóloga , Humanos , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína de Replicación A/metabolismoRESUMEN
Glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumor. Standard treatment for GBM patients is surgery followed by radiotherapy and/or chemotherapy, but tumors always recur. Traditional therapies seem to fail because they eliminate only the bulk of the tumors and spare a population of stem-like cells termed tumor-initiating cells. The stem-like state and preferential activation of DNA damage response in the GBM tumor-initiating cells contribute to their radio-resistance and recurrence. The molecular mechanisms underlying this efficient activation of damage response and maintenance of stem-like state remain elusive. Here we show that RBM14 controls DNA repair pathways and also prevents cell differentiation in GBM spheres, causing radio-resistance. Knockdown of RBM14 affects GBM sphere maintenance and sensitizes radio-resistant GBM cells at the cellular level. We demonstrate that RBM14 knockdown blocks GBM regrowth after irradiation in vivo. In addition, RBM14 stimulates DNA repair by controlling the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway. These results reveal unexpected functions of the RNA-binding protein RBM14 in control of DNA repair and maintenance of tumor-initiating cells. Targeting the RBM14-dependent pathway may prevent recurrence of tumors and eradicate the deadly disease completely.
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Neoplasias Encefálicas/patología , Diferenciación Celular/efectos de la radiación , Reparación del ADN/efectos de la radiación , Glioblastoma/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Madre Neoplásicas/patología , Tolerancia a Radiación , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Ciclo Celular , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Rayos gamma , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/radioterapia , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de la radiación , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DNA damage occurs during DNA replication, spontaneous chemical reactions, and assaults by external or metabolism-derived agents. Therefore, all living cells must constantly contend with DNA damage. Cells protect themselves from these genotoxic stresses by activating the DNA damage checkpoint and DNA repair pathways. Coordination of these pathways requires tight regulation in order to prevent genomic instability. The checkpoint clamp complex consists of Rad9, Rad1 and Hus1 proteins, and is often called the 9-1-1 complex. This PCNA (proliferating cell nuclear antigen)-like donut-shaped protein complex is a checkpoint sensor protein that is recruited to DNA damage sites during the early stage of the response, and is required for checkpoint activation. As PCNA is required for multiple pathways of DNA metabolism, the checkpoint clamp has also been implicated in direct roles in DNA repair, as well as in coordination of the pathways. Here we discuss roles of the checkpoint clamp in DNA damage response (DDR).
RESUMEN
Upon genotoxic stress and during normal S phase, ATM phosphorylates the checkpoint clamp protein Rad9 in a manner that depends on Ser272. Ser272 is the only known ATM-dependent phosphorylation site in human Rad9. However, Ser272 phosphorylation is not required for survival or checkpoint activation after DNA damage. The physiological function of Ser272 remains elusive. Here, we show that ATM-dependent Rad9(Ser272) phosphorylation requires the MRN complex and controls repair pathways. Furthermore, the mutant cells accumulate large numbers of chromosome breaks and induce gross chromosomal rearrangements. Our findings establish a new and unexpected role for ATM: it phosphorylates the checkpoint clamp in order to control repair pathways, thereby maintaining genomic integrity during unperturbed cell cycle and upon DNA damage.
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Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ácido Anhídrido Hidrolasas , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Rotura Cromosómica , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Proteína Homóloga de MRE11 , Morfolinas/farmacología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxidación-Reducción , Fosforilación , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Pironas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Reparación del ADN por Recombinación , Serina/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
When replication forks collapse, Rad3 phosphorylates the checkpoint-clamp protein Rad9 in a manner that depends on Thr 225, a residue within the PCNA-like domain. The physiological function of Thr 225-dependent Rad9 phosphorylation, however, remains elusive. Here, we show that Thr 225-dependent Rad9 phosphorylation by Rad3 regulates DNA repair pathways. A rad9(T225C) mutant induces a translesion synthesis (TLS)-dependent high spontaneous mutation rate and a hyper-recombination phenotype. Consistent with this, Rad9 coprecipitates with the post-replication repair protein Mms2. This interaction is dependent on Rad9 Thr 225 and is enhanced by DNA damage. Genetic analyses indicate that Thr 225-dependent Rad9 phosphorylation prevents inappropriate Rhp51-dependent recombination, potentially by redirecting the repair through a Pli1-mediated sumoylation pathway into the error-free branch of the Rhp6 repair pathway. Our findings reveal a new mechanism by which phosphorylation of Rad9 at Thr 225 regulates the choice of repair pathways for maintaining genomic integrity during the cell cycle.
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Proteínas de Ciclo Celular/metabolismo , Reparación del ADN/genética , Genes cdc/fisiología , Inestabilidad Genómica/genética , Proteínas Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2 , Daño del ADN/genética , Regulación Fúngica de la Expresión Génica/genética , Fosforilación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transducción de Señal/genética , Treonina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Mutations in genome caretaker genes can induce genomic instability, which are potentially early events in tumorigenesis. Cells have evolved biological processes to cope with the genomic insults. One is a multifaceted response, termed checkpoint, which is a network of signaling pathways to coordinate cell cycle transition with DNA repair, activation of transcriptional programs, and induction of tolerance of the genomic perturbations. When genomic perturbations are beyond repair, checkpoint responses can also induce apoptosis or senescence to eliminate those deleterious damaged cells. Fission yeast, Schizosaccharomyces pombe (S. pombe) has served as a valuable model organism for studies of the checkpoint signaling pathways. In this chapter, we describe methods used to analyze mutagenesis and recombinational repair induced by genomic perturbations, and methods used to detect the checkpoint responses to replication stress and DNA damage in fission yeast cells. In the first section, we present methods used to analyze the mutation rate, mutation spectra, and recombinational repair in fission yeast when replication is perturbed by either genotoxic agents or mutations in genomic caretaker gene such as DNA replication genes. In the second section, we describe methods used to examine checkpoint activation in response to chromosome replication stress and DNA damage. In the final section, we comment on how checkpoint activation regulates mutagenic synthesis by a translesion DNA polymerase in generating a mutator phenotype of small sequence alterations in cells, and how a checkpoint kinase appropriately regulates an endonuclease complex to either prevent or allow deletion of genomic sequences and recombinational repair when fission yeast cells experience genomic perturbation in order to avoid deleterious mutations and maintain cell growth.
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Mutagénesis , Schizosaccharomyces/genética , Daño del ADN , Replicación del ADN , Recombinación GenéticaRESUMEN
The replication checkpoint kinase Cds1 preserves genome integrity by stabilizing stalled replication forks. Cds1 targets substrates through its FHA domain. The Cds1 FHA domain interacts with Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease. We report here that Mus81 and Rhp51 are required for generating deletion mutations in fission yeast replication mutants that experience replication stress. A mutation in the Mus81 FHA-binding motif eliminates its Cds1-binding and Cds1-dependent phosphorylation. Furthermore, this mutation exacerbates the deletion mutator phenotype of a replication mutant, and induces a hyper-recombination phenotype in hydroxyurea-treated cells. In unperturbed cells, Mus81 associates with chromatin throughout S phase. In replication mutants grown at semipermissive temperature, Mus81 undergoes minor Cds1-dependent phosphorylation, remains chromatin-associated, generates deletion mutations, and maintains cell growth. Upon S-phase arrest by acute hydroxyurea treatment, Mus81 is not required for cell viability but is essential for recovery from replication fork collapse. Moreover, Mus81 undergoes extensive Cds1-dependent phosphorylation and dissociates from chromatin in hydroxyurea-arrested cells, thereby preventing it from cleaving stalled replication forks that could lead to fork breakage and chromosomal rearrangement. These results provide novel insights into how Cds1 regulates Mus81 accordingly when cells experience different replication stress to preserve genome integrity.
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Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Inestabilidad Genómica/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Quinasa de Punto de Control 2 , Cromatina/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Hidroxiurea/toxicidad , Mutación/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Recombinasa Rad51 , Recombinación Genética/genética , Fase S/efectos de los fármacos , Fase S/fisiología , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polkappa). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.
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Ciclo Celular , Reparación del ADN/fisiología , Replicación del ADN , Mutagénesis , Schizosaccharomyces/genética , Transducción de Señal , Proteínas de Ciclo Celular/fisiología , Schizosaccharomyces/citologíaRESUMEN
Cells have evolved checkpoint responses to arrest or delay the cell cycle, activate DNA repair networks, or induce apoptosis after genomic perturbation. Cells have also evolved the translesion synthesis processes to tolerate genomic lesions by either error-free or error-prone repair. Here, we show that after a replication perturbation, cells exhibit a mutator phenotype, which can be significantly affected by mutations in the checkpoint elements Cds1 and Rad17 or translesion synthesis polymerases DinB and Polzeta. Cells respond to genomic perturbation by up-regulation of DinB in a checkpoint activation-dependent manner. Moreover, association of DinB with chromatin is dependent on functional Rad17, and DinB physically interacts with the checkpoint-clamp components Hus1 and Rad1. Thus, translesion synthesis is a part of the checkpoint response.