RESUMEN
Telomerase, the enzyme that maintains telomeres at natural chromosome ends, should be repressed at double-strand breaks (DSBs), where neotelomere formation can cause terminal truncations. We developed an assay to detect neotelomere formation at Cas9- or I-SceI-induced DSBs in human cells. Telomerase added telomeric repeats to DSBs, leading to interstitial telomeric repeat insertions or the formation of functional neotelomeres accompanied by terminal deletions. The threat that telomerase poses to genome integrity was minimized by ataxia telangiectasia and Rad3-related (ATR) kinase signaling, which inhibited telomerase at resected DSBs. In addition to acting at resected DSBs, telomerase used the extruded strand in the Cas9 enzyme-product complex as a primer for neotelomere formation. We propose that although neotelomere formation is detrimental in normal human cells, it may allow cancer cells to escape from breakage-fusion-bridge cycles.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Roturas del ADN de Doble Cadena , Telomerasa , Telómero , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Telomerasa/genética , Telómero/genética , Telómero/metabolismo , Técnicas Genéticas , Proteína 9 Asociada a CRISPR , Células HeLaRESUMEN
Short-read sequencing is the workhorse of cancer genomics yet is thought to miss many structural variants (SVs), particularly large chromosomal alterations. To characterize missing SVs in short-read whole genomes, we analyzed 'loose ends'-local violations of mass balance between adjacent DNA segments. In the landscape of loose ends across 1,330 high-purity cancer whole genomes, most large (>10-kb) clonal SVs were fully resolved by short reads in the 87% of the human genome where copy number could be reliably measured. Some loose ends represent neotelomeres, which we propose as a hallmark of the alternative lengthening of telomeres phenotype. These pan-cancer findings were confirmed by long-molecule profiles of 38 breast cancer and melanoma cases. Our results indicate that aberrant homologous recombination is unlikely to drive the majority of large cancer SVs. Furthermore, analysis of mass balance in short-read whole genome data provides a surprisingly complete picture of cancer chromosomal structure.
Asunto(s)
Neoplasias de la Mama , Genómica , Humanos , Femenino , Genómica/métodos , Análisis de Secuencia de ADN/métodos , Genoma Humano/genética , Aberraciones Cromosómicas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Variación Estructural del Genoma/genéticaRESUMEN
Telomeres replicated by leading-strand synthesis lack the 3' overhang required for telomere protection. Surprisingly, resection of these blunt telomeres is initiated by the telomere-specific 5' exonuclease Apollo rather than the Mre11-Rad50-Nbs1 (MRN) complex, the nuclease that acts at DNA breaks. Without Apollo, leading-end telomeres undergo fusion, which, as demonstrated here, is mediated by alternative end joining. Here, we show that DNA-PK and TRF2 coordinate the repression of MRN at blunt mouse telomeres. DNA-PK represses an MRN-dependent long-range resection, while the endonuclease activity of MRN-CtIP, which could cleave DNA-PK off of blunt telomere ends, is inhibited in vitro and in vivo by the iDDR of TRF2. AlphaFold-Multimer predicts a conserved association of the iDDR with Rad50, potentially interfering with CtIP binding and MRN endonuclease activation. We propose that repression of MRN-mediated resection is a conserved aspect of telomere maintenance and represents an ancient feature of DNA-PK and the iDDR.
Asunto(s)
Roturas del ADN , Proteína Quinasa Activada por ADN , Animales , Ratones , Endonucleasas , Telómero , ADNRESUMEN
Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of experimental telomere crisis identified in previous studies. We examine the spectrum of structural variants (SVs) instigated by natural telomere crisis. Eight spontaneous post-crisis clones did not show prominent patterns of BFB cycles or chromothripsis. Their crisis-induced genome rearrangements varied from infrequent simple SVs to more frequent and complex SVs. In contrast, BFB cycles and chromothripsis occurred in MRC5 fibroblast clones that escaped telomere crisis after CRISPR-controlled telomerase activation. This system revealed convergent evolutionary lineages altering one allele of chromosome 12p, where a short telomere likely predisposed to fusion. Remarkably, the 12p chromothripsis and BFB events were stabilized by independent fusions to chromosome 21. The data establish that telomere crisis can generate a wide spectrum of SVs implying that a lack of BFB patterns and chromothripsis in cancer genomes does not indicate absence of past telomere crisis.
Asunto(s)
Cromotripsis , Neoplasias/genética , Telómero/química , Línea Celular , Inestabilidad Cromosómica , Fibroblastos , Genoma , Inestabilidad Genómica , Humanos , Pulmón , Metafase , Modelos Biológicos , Telómero/ultraestructuraRESUMEN
Telomere shortening is a presumed tumor suppressor pathway that imposes a proliferative barrier (the Hayflick limit) during tumorigenesis. This model predicts that excessively long somatic telomeres predispose to cancer. Here, we describe cancer-prone families with two unique TINF2 mutations that truncate TIN2, a shelterin subunit that controls telomere length. Patient lymphocyte telomeres were unusually long. We show that the truncated TIN2 proteins do not localize to telomeres, suggesting that the mutations create loss-of-function alleles. Heterozygous knock-in of the mutations or deletion of one copy of TINF2 resulted in excessive telomere elongation in clonal lines, indicating that TINF2 is haploinsufficient for telomere length control. In contrast, telomere protection and genome stability were maintained in all heterozygous clones. The data establish that the TINF2 truncations predispose to a tumor syndrome. We conclude that TINF2 acts as a haploinsufficient tumor suppressor that limits telomere length to ensure a timely Hayflick limit.
Asunto(s)
Genes Supresores de Tumor , Acortamiento del Telómero/genética , Proteínas de Unión a Telómeros/fisiología , Telómero/genética , Línea Celular , Femenino , Células HEK293 , Heterocigoto , Humanos , Mutación con Pérdida de Función , Masculino , Neoplasias/genética , Telómero/patología , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteínas Supresoras de TumorRESUMEN
TRF1 facilitates the replication of telomeric DNA in part by recruiting the BLM helicase, which can resolve G-quadruplexes on the lagging-strand template. Lagging-strand telomeres lacking TRF1 or BLM form fragile telomeres-structures that resemble common fragile sites (CFSs)-but how they are formed is not known. We report that analogous to CFSs, fragile telomeres in BLM-deficient cells involved double-strand break (DSB) formation, in this case by the SLX4/SLX1 nuclease. The DSBs were repaired by POLD3/POLD4-dependent break-induced replication (BIR), resulting in fragile telomeres containing conservatively replicated DNA. BIR also promoted fragile telomere formation in cells with FokI-induced telomeric DSBs and in alternative lengthening of telomeres (ALT) cells, which have spontaneous telomeric damage. BIR of telomeric DSBs competed with PARP1-, LIG3-, and XPF-dependent alternative nonhomologous end joining (alt-NHEJ), which did not generate fragile telomeres. Collectively, these findings indicate that fragile telomeres can arise from BIR-mediated repair of telomeric DSBs.
Asunto(s)
Sitios Frágiles del Cromosoma/genética , Roturas del ADN de Doble Cadena , Replicación del ADN , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Telómero/patología , Animales , Línea Celular , Células Cultivadas , Reparación del ADN , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Fibroblastos , Humanos , Ratones , Recombinasas/genética , Recombinasas/metabolismoRESUMEN
Alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler (ATRX), a DAXX (death domain-associated protein) interacting protein, is often lost in cells using the alternative lengthening of telomeres (ALT) pathway, but it is not known how ATRX loss leads to ALT. We report that ATRX deletion from mouse cells altered the repair of telomeric double-strand breaks (DSBs) and induced ALT-like phenotypes, including ALT-associated promyelocytic leukemia (PML) bodies (APBs), telomere sister chromatid exchanges (T-SCEs), and extrachromosomal telomeric signals (ECTSs). Mechanistically, we show that ATRX affects telomeric DSB repair by promoting cohesion of sister telomeres and that loss of ATRX in ALT cells results in diminished telomere cohesion. In addition, we document a role for DAXX in the repair of telomeric DSBs. Removal of telomeric cohesion in combination with DAXX deficiency recapitulates all telomeric DSB repair phenotypes associated with ATRX loss. The data reveal that ATRX has an effect on telomeric DSB repair and that this role involves both telomere cohesion and a DAXX-dependent pathway.
Asunto(s)
Proteínas Co-Represoras/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Chaperonas Moleculares/fisiología , Intercambio de Cromátides Hermanas/genética , Telómero/genética , Proteína Nuclear Ligada al Cromosoma X/fisiología , Animales , Células Cultivadas , Embrión de Mamíferos , Femenino , Células HeLa , Humanos , Masculino , Discapacidad Intelectual Ligada al Cromosoma X/genética , Discapacidad Intelectual Ligada al Cromosoma X/patología , Ratones , Ratones Noqueados , Transducción de Señal/genética , Telómero/metabolismo , Homeostasis del Telómero/genética , Talasemia alfa/genética , Talasemia alfa/patologíaRESUMEN
Circulating tumor cells (CTCs) are potential precursors of metastatic cancer, and genomic information obtained from CTCs have the potential to provide new insights into the biology of cancer metastasis. We previously developed a technique for single-cell manipulation based on the encapsulation of a single cell in a photopolymerized hydrogel that can be used for subsequent genetic analysis. However, this technique has limitations in terms of throughput because light irradiation must be performed on each individual cell from the confocal laser-scanning microscopy. Here, we present a high-throughput cell manipulation technique using a multiple single-cell encapsulation system with a digital micromirror device. This system enables rapid cell imaging within a microcavity array, a microfilter for the recovery of CTCs from blood samples, as well as the simultaneous encapsulation of several CTCs with hydrogels photopolymerized using a multiple light-irradiation system. Furthermore, single-cell labeling using two differently shaped hydrogels was examined to distinguish between NCI-H1975 cells and A549 cells, demonstrating the utility of the system for single-cell gene mutation analysis. In addition to CTCs, our system can be widely applied for analyses of mammalian cells and microorganisms.
RESUMEN
In DNA repair, the resection of double-strand breaks dictates the choice between homology-directed repair-which requires a 3' overhang-and classical non-homologous end joining, which can join unresected ends1,2. BRCA1-mutant cancers show minimal resection of double-strand breaks, which renders them deficient in homology-directed repair and sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1)3-8. When BRCA1 is absent, the resection of double-strand breaks is thought to be prevented by 53BP1, RIF1 and the REV7-SHLD1-SHLD2-SHLD3 (shieldin) complex, and loss of these factors diminishes sensitivity to PARP1 inhibitors4,6-9. Here we address the mechanism by which 53BP1-RIF1-shieldin regulates the generation of recombinogenic 3' overhangs. We report that CTC1-STN1-TEN1 (CST)10, a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)-primase11, is a downstream effector in the 53BP1 pathway. CST interacts with shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and shieldin-dependent manner. As with loss of 53BP1, RIF1 or shieldin, the depletion of CST leads to increased resection. In BRCA1-deficient cells, CST blocks RAD51 loading and promotes the efficacy of PARP1 inhibitors. In addition, Polα inhibition diminishes the effect of PARP1 inhibitors. These data suggest that CST-Polα-mediated fill-in helps to control the repair of double-strand breaks by 53BP1, RIF1 and shieldin.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Polimerasa I/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Proteína BRCA1/deficiencia , Línea Celular , ADN Primasa/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Humanos , Ratones , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Reparación del ADN por Recombinación , Telómero/genética , Telómero/metabolismoRESUMEN
Circulating tumor cells (CTCs) provide potentially accessible in vivo sources of metastatic cancer cells. As such, considerable focus has been placed on analyzing the genetics of single-CTCs. Prior to these analyses, however, CTCs must first be detected within the blood samples of cancer patients. Current methods for detection of CTCs by fluorescence microscopy require the analysis of hundreds of images per blood sample, making this a time-consuming process that creates a bottleneck in CTC analysis. In this study, we therefore developed a wide-field fluorescence imaging system for rapid CTC detection. For these analyses, CTCs were first isolated using the microcavity array (MCA), a micro-sized filter for CTC recovery that separates cells based on differences in cell size and deformability. Notably, the proposed imaging system enabled rapid (â¼10 s) visualization of all stained cells within the 6 mm × 6 mm MCA field via one-shot imaging. Furthermore, the morphology of the cells in the resulting images accurately reflected that observed by conventional microscopy. In total, isolation and detection of CTCs using the MCA combined with our novel wide-field fluorescence imaging system was achieved within 1 h. Thus, our proposed system will provide rapid CTC detection system.
Asunto(s)
Microscopía Fluorescente , Células Neoplásicas Circulantes , Imagen Óptica , HumanosRESUMEN
YTH domain containing 2 (YTHDC2) is a member of the DExD/H-box family of ATP-dependent RNA helicases. We previously found that YTHDC2 expression is up-regulated in several human cancer cells. In this study, we demonstrate novel roles for YTHDC2 in metastasis of colon tumor cells via translation-dependent pathway. Knockdown of YTHDC2 attenuated protein expression of metastasis-related genes, such as hypoxia-inducible factor-1alpha (HIF-1α), and inhibited metastasis of colon tumor cells in vitro and in vivo. To confirm that YTHDC2 promotes translation initiation by unwinding the 5'-untranslated region (5'UTR) of mRNA, we constructed a firefly luciferase reporter containing the 5'UTR of the HIF-1α mRNA and showed reduction in luciferase activity in YTHDC2-silenced cells. Furthermore, we examined expression levels of YTHDC2 by immunohistochemical staining in human colon cancer tissues from 72 patients and found a significantly positive correlation between YTHDC2 expression and the tumor stage, including metastasis. In conclusion, these results suggest that the RNA helicase YTHDC2 contributes to colon tumor metastasis by promoting translation of HIF-1α and that YTHDC2 is potentially a diagnostic marker and target gene for treating colon cancer patients.
Asunto(s)
Adenocarcinoma/enzimología , Adenosina Trifosfatasas/metabolismo , Movimiento Celular , Neoplasias del Colon/enzimología , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , ARN Mensajero/metabolismo , Regiones no Traducidas 5' , Adenocarcinoma/genética , Adenocarcinoma/secundario , Adenosina Trifosfatasas/genética , Animales , Células COS , Chlorocebus aethiops , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Células HCT116 , Células HT29 , Xenoinjertos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Trasplante de Neoplasias , Proteínas Nucleares/metabolismo , ARN Helicasas , Interferencia de ARN , ARN Mensajero/genética , Transducción de Señal , Factores de Tiempo , Transfección , Hipoxia Tumoral , Proteína 1 Relacionada con Twist/metabolismo , Regulación hacia ArribaRESUMEN
Materials with reduced dimensionality have attracted much interest in various fields of fundamental and applied science. True one-dimensional (1D) crystals with single-atom thickness have been realized only for few elemental metals (Au, Ag) or carbon, all of which showed very short lifetimes under ambient conditions. We demonstrate here a successful synthesis of stable 1D ionic crystals in which two chemical elements, one being a cation and the other an anion, align alternately inside carbon nanotubes. Unusual dynamical behaviours for different atoms in the 1D lattice are experimentally corroborated and suggest substantial interactions of the atoms with the nanotube sheath. Our theoretical studies indicate that the 1D ionic crystals have optical properties distinct from those of their bulk counterparts and that the properties can be engineered by introducing atomic defects into the chains.
RESUMEN
Chemically converted graphene (CCG) covalently linked with porphyrins has been prepared by a Suzuki coupling reaction between iodophenyl-functionalized CCG and porphyrin boronic ester. The covalently linked CCG-porphyrin composite was designed to possess a short, rigid phenylene spacer between the porphyrin and the CCG. The composite material formed stable dispersions in DMF and the structure was characterized by spectroscopic, thermal, and microscopic measurements. In steady-state photoluminescence spectra, the emission from the porphyrin linked to the CCG was quenched strongly relative to that of the porphyrin reference. Fluorescence lifetime and femtosecond transient absorption measurements of the porphyrin-linked CCG revealed a short-lived porphyrin singlet excited state (38 ps) without yielding the porphyrin radical cation, thereby substantiating the occurrence of energy transfer from the porphyrin excited state to the CCG and subsequent rapid decay of the CCG excited state to the ground state. Consistently, the photocurrent action spectrum of a photoelectrochemical device with a SnO(2) electrode coated with the porphyrin-linked CCG exhibited no photocurrent response from the porphyrin absorption. The results obtained here provide deep insight into the interaction between graphenes and π-conjugated systems in the excited and ground states.
RESUMEN
To prevent ATR activation, telomeres deploy the single-stranded DNA binding activity of TPP1/POT1a. POT1a blocks the binding of RPA to telomeres, suggesting that ATR is repressed through RPA exclusion. However, comparison of the DNA binding affinities and abundance of TPP1/POT1a and RPA indicates that TPP1/POT1a by itself is unlikely to exclude RPA. We therefore analyzed the central shelterin protein TIN2, which links TPP1/POT1a (and POT1b) to TRF1 and TRF2 on the double-stranded telomeric DNA. Upon TIN2 deletion, telomeres lost TPP1/POT1a, accumulated RPA, elicited an ATR signal, and showed all other phenotypes of POT1a/b deletion. TIN2 also affected the TRF2-dependent repression of ATM kinase signaling but not to TRF2-mediated inhibition of telomere fusions. Thus, while TIN2 has a minor contribution to the repression of ATM by TRF2, its major role is to stabilize TPP1/POT1a on the ss telomeric DNA, thereby allowing effective exclusion of RPA and repression of ATR signaling.
Asunto(s)
Reparación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Transducción de Señal/genética , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Ratones , Ratones Noqueados , Unión Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
The molecular orientation of ellipsoidal C(70) in single-walled carbon nanotubes (SWCNTs) depends on the tube diameter (d(t)). Photoluminescence (PL) studies reveal that the fullerene encapsulation effects on the optical transition energy of SWCNTs are significantly different for C(70) and C(60) at d(t) = 1.405-1.431 nm. This indicates that the transition from the "lying" alignment to the "standing" alignment occurs at d(t) ≈ 1.41 nm and the electronic states of SWCNTs are very sensitive to the interspacing between the encapsulated molecules and the SWCNTs. The present findings suggest that the electronic structure of SWCNTs is tunable not only by alternating the encapsulated molecules but also by controlling their molecular orientations, thus paving the way for development of novel SWCNT-based devices.
RESUMEN
Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1.POT1 heterodimer was present 50-100 copies/telomere, which is in excess of its single-stranded telomeric DNA binding sites, indicating that some of the TPP1.POT1 in shelterin is not associated with the single-stranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were approximately 10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1 x POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.
Asunto(s)
Complejos Multiproteicos/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Animales , Daño del ADN/fisiología , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Células HeLa , Humanos , Ratones , Complejos Multiproteicos/genética , Células 3T3 NIH , Complejo Shelterina , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Although Tel2 was identified as a budding yeast gene required for the telomere length maintenance, we found no obvious telomeric function for mammalian Tel2. Targeted gene deletion showed that mouse Tel2 is essential in embryonic development, embryonic stem (ES) cells, and embryonic fibroblasts. Conditional deletion of Tel2 from embryonic fibroblasts compromised their response to IR and UV, diminishing the activation of checkpoint kinases and their downstream effectors. The effects of Tel2 deletion correlated with significantly reduced protein levels for the PI3K-related kinases ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), DNA-dependent protein kinase catalytic subunit ataxia (DNA-PKcs). Tel2 deletion also elicited specific depletion of the mammalian target of rapamycin (mTOR), suppressor with morphological effect on genitalia 1 (SMG1), and transformation/transcription domain-associated protein (TRRAP), and curbed mTOR signaling, indicating that Tel2 affects all six mammalian PIKKs. While Tel2 deletion did not alter PIKK mRNA levels, in vivo pulse labeling experiments showed that Tel2 controls the stability of ATM and mTOR. Each of the PIKK family members associated with Tel2 in vivo and in vitro experiments indicated that Tel2 binds to part of the HEAT repeat segments of ATM and mTOR. These data identify Tel2 as a highly conserved regulator of PIKK stability.
Asunto(s)
Fibroblastos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Proteínas de Unión a Telómeros/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Daño del ADN , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Embrión de Mamíferos , Fibroblastos/enzimología , Fibroblastos/efectos de la radiación , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genotipo , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/metabolismo , Fenotipo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Serina-Treonina Quinasas TOR , Proteínas de Unión a Telómeros/genética , Transfección , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The shelterin complex at mammalian telomeres contains the single-stranded DNA-binding protein Pot1, which regulates telomere length and protects chromosome ends. Pot1 binds Tpp1, the shelterin component that connects Pot1 to the duplex telomeric DNA-binding proteins Trf1 and Trf2. Control of telomere length requires that Pot1 binds Tpp1 as well as the single-stranded telomeric DNA, but it is not known whether the protective function of Pot1 depends on Tpp1. Alternatively, Pot1 might function similarly to the Pot1-like proteins of budding and fission yeast, which have no known Tpp1-like connection to the duplex telomeric DNA. Using mutant mouse cells with diminished Tpp1 levels, RNA interference directed to mouse Tpp1 and Pot1, and complementation of mouse Pot1 knockout cells with human and mouse Pot1 variants, we show here that Tpp1 is required for the protective function of mammalian Pot1 proteins.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/fisiología , Telómero/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/fisiología , Interferencia de ARN , Complejo Shelterina , Proteínas Similares a la Proteína de Unión a TATA-Box/antagonistas & inhibidores , Proteínas Similares a la Proteína de Unión a TATA-Box/fisiología , Proteínas de Unión a Telómeros/genética , Proteína 2 de Unión a Repeticiones TeloméricasRESUMEN
ATM and rad3-related protein kinase (ATR), a member of the phosphoinositide kinase-like protein kinase family, plays a critical role in cellular responses to DNA structural abnormalities in conjunction with its interacting protein, ATRIP. Here, we show that the amino-terminal portion of ATRIP is relocalized to DNA damage-induced nuclear foci in an RPA-dependent manner, despite its lack of ability to associate with ATR. In addition, ATR-free ATRIP protein can be recruited to the nuclear foci. Our results suggest that the N-terminal domain of the ATRIP protein contributes to the cell cycle checkpoint by regulating the intranuclear localization of ATR.