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1.
Annu Rev Biochem ; 83: 519-52, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24606147

RESUMEN

RecQ helicases are an important family of genome surveillance proteins conserved from bacteria to humans. Each of the five human RecQ helicases plays critical roles in genome maintenance and stability, and the RecQ protein family members are often referred to as guardians of the genome. The importance of these proteins in cellular homeostasis is underscored by the fact that defects in BLM, WRN, and RECQL4 are linked to distinct heritable human disease syndromes. Each human RecQ helicase has a unique set of protein-interacting partners, and these interactions dictate its specialized functions in genome maintenance, including DNA repair, recombination, replication, and transcription. Human RecQ helicases also interact with each other, and these interactions have significant impact on enzyme function. Future research goals in this field include a better understanding of the division of labor among the human RecQ helicases and learning how human RecQ helicases collaborate and cooperate to enhance genome stability.


Asunto(s)
Reparación del ADN , Replicación del ADN , RecQ Helicasas/fisiología , Recombinación Genética , ADN/química , Exodesoxirribonucleasas/química , Genoma Humano , Inestabilidad Genómica , Humanos , Modelos Moleculares , Conformación Molecular , Familia de Multigenes , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , RecQ Helicasas/química , Fase S , Helicasa del Síndrome de Werner
2.
Mol Cell ; 75(1): 117-130.e6, 2019 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-31101499

RESUMEN

Telomeres are essential for genome stability. Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere shortening. Although telomeres are hypersensitive to ROS-mediated 8-oxoguanine (8-oxoG) formation, the biological effect of this common lesion at telomeres is poorly understood because ROS have pleiotropic effects. Here we developed a chemoptogenetic tool that selectively produces 8-oxoG only at telomeres. Acute telomeric 8-oxoG formation increased telomere fragility in cells lacking OGG1, the enzyme that removes 8-oxoG, but did not compromise cell survival. However, chronic telomeric 8-oxoG induction over time shortens telomeres and impairs cell growth. Accumulation of telomeric 8-oxoG in chronically exposed OGG1-deficient cells triggers replication stress, as evidenced by mitotic DNA synthesis at telomeres, and significantly increases telomere losses. These losses generate chromosome fusions, leading to chromatin bridges and micronucleus formation upon cell division. By confining base damage to the telomeres, we show that telomeric 8-oxoG accumulation directly drives telomere crisis.


Asunto(s)
Aberraciones Cromosómicas/efectos de la radiación , ADN Glicosilasas/genética , Reparación del ADN/efectos de la radiación , Inestabilidad Genómica/efectos de la radiación , Guanina/análogos & derivados , Telómero/efectos de la radiación , División Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Daño del ADN , ADN Glicosilasas/deficiencia , Replicación del ADN/efectos de la radiación , Expresión Génica , Guanina/agonistas , Guanina/biosíntesis , Células HeLa , Humanos , Luz/efectos adversos , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Optogenética , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoblastos/efectos de la radiación , Estrés Oxidativo/efectos de la radiación , Oxígeno Singlete/agonistas , Oxígeno Singlete/metabolismo , Telómero/metabolismo , Homeostasis del Telómero/efectos de la radiación
3.
Trends Genet ; 39(8): 593-595, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37179160

RESUMEN

Telomeres are transcribed into long noncoding telomeric repeat-containing RNA (TERRA). Or so we thought. Recently, Al-Turki and Griffith provided evidence that TERRA can code for valine-arginine (VR) or glycine-leucine (GL) dipeptide repeat proteins by undergoing repeat-associated non-ATG (RAN) translation. This finding uncovers a new mechanism by which telomeres can impact cellular function.


Asunto(s)
ARN Largo no Codificante , ARN Largo no Codificante/genética , Telómero/genética , Telómero/metabolismo
4.
Nucleic Acids Res ; 52(17): 10490-10503, 2024 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-39189448

RESUMEN

Telomeric repeat containing RNA (TERRA) is a noncoding RNA that is transcribed from telomeres. Previous study showed that TERRA trans anneals by invading into the telomeric duplex to form an R-loop in mammalian cells. Here, we elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins, RAD51 and RNase H using single molecule (sm) assays. We demonstrate that TERRA trans annealing into telomeric DNA exhibits dynamic movement that is stabilized by TRF2. TERRA annealing to the telomeric duplex results in the formation of a stable triplex structure which differs from a conventional R-loop. We identified that the presence of a sub-telomeric DNA and a telomeric overhang in the form of a G-quadruplex significantly enhances TERRA annealing to telomeric duplex. We also demonstrate that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Additionally, TRF2 increases TERRA affinity to telomeric duplex and protects it from RNase H digestion. In contrast, TRF1 represses TERRA annealing to telomeric duplex and fails to provide protection against RNase H digestion. Our findings provide an in-depth molecular mechanism underpinning TERRA recruitment and annealing to the telomere.


Asunto(s)
Recombinasa Rad51 , Ribonucleasa H , Telómero , Proteína 1 de Unión a Repeticiones Teloméricas , Proteína 2 de Unión a Repeticiones Teloméricas , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Ribonucleasa H/metabolismo , Recombinasa Rad51/metabolismo , Humanos , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/genética , G-Cuádruplex , ADN/metabolismo , ADN/química , ADN/genética , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/genética , Estructuras R-Loop , ARN no Traducido/metabolismo , ARN no Traducido/genética , ARN no Traducido/química , Complejo Shelterina/metabolismo , Imagen Individual de Molécula
5.
Nucleic Acids Res ; 52(4): 1763-1778, 2024 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-38153143

RESUMEN

BG4 is a single-chain variable fragment antibody shown to bind various G-quadruplex (GQ) topologies with high affinity and specificity, and to detect GQ in cells, including GQ structures formed within telomeric TTAGGG repeats. Here, we used ELISA and single-molecule pull-down (SiMPull) detection to test how various lengths and GQ destabilizing base modifications in telomeric DNA constructs alter BG4 binding. We observed high-affinity BG4 binding to telomeric GQ independent of telomere length, although three telomeric repeat constructs that cannot form stable intramolecular GQ showed reduced affinity. A single guanine substitution with 8-aza-7-deaza-G, T, A, or C reduced affinity to varying degrees depending on the location and base type, whereas two G substitutions in the telomeric construct dramatically reduced or abolished binding. Substitution with damaged bases 8-oxoguanine and O6-methylguanine failed to prevent BG4 binding although affinity was reduced depending on lesion location. SiMPull combined with FRET revealed that BG4 binding promotes folding of telomeric GQ harboring a G to T substitution or 8-oxoguanine. Atomic force microscopy revealed that BG4 binds telomeric GQ with a 1:1 stoichiometry. Collectively, our data suggest that BG4 can recognize partially folded telomeric GQ structures and promote telomeric GQ stability.


Asunto(s)
G-Cuádruplex , ADN/genética , ADN/química , Telómero/genética , Anticuerpos/genética
6.
Proc Natl Acad Sci U S A ; 119(51): e2211775119, 2022 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-36508676

RESUMEN

Synthetic lethality is a powerful approach for targeting oncogenic drivers in cancer. Recent studies revealed that cancer cells with microsatellite instability (MSI) require Werner (WRN) helicase for survival; however, the underlying mechanism remains unclear. In this study, we found that WRN depletion strongly induced p53 and its downstream apoptotic target PUMA in MSI colorectal cancer (CRC) cells. p53 or PUMA deletion abolished apoptosis induced by WRN depletion in MSI CRC cells. Importantly, correction of MSI abrogated the activation of p53/PUMA and cell killing, while induction of MSI led to sensitivity in isogenic CRC cells. Rare p53-mutant MSI CRC cells are resistant to WRN depletion due to lack of PUMA induction, which could be restored by wildtype (WT) p53 knock in or reconstitution. WRN depletion or treatment with the RecQ helicase inhibitor ML216 suppressed in vitro and in vivo growth of MSI CRCs in a p53/PUMA-dependent manner. ML216 treatment was efficacious in MSI CRC patient-derived xenografts. Interestingly, p53 gene remains WT in the majority of MSI CRCs. These results indicate a critical role of p53/PUMA-mediated apoptosis in the vulnerability of MSI CRCs to WRN loss, and support WRN as a promising therapeutic target in p53-WT MSI CRCs.


Asunto(s)
Neoplasias del Colon , Neoplasias Colorrectales , Humanos , Helicasa del Síndrome de Werner/genética , Proteína p53 Supresora de Tumor/genética , Inestabilidad de Microsatélites , Neoplasias Colorrectales/genética , RecQ Helicasas/genética
7.
Nucleic Acids Res ; 50(11): 6271-6283, 2022 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-35687089

RESUMEN

Human telomere overhang composed of tandem repeats of TTAGGG folds into G-quadruplex (G4). Unlike in an experimental setting in the test tube in which the entire length is allowed to fold at once, inside the cell, the overhang is expected to fold as it is synthesized directionally (5' to 3') and released segmentally by a specialized enzyme, the telomerase. To mimic such vectorial G4 folding process, we employed a superhelicase, Rep-X which can unwind DNA to release the TTAGGG repeats in 5' to 3' direction. We demonstrate that the folded conformation achieved by the refolding of full sequence is significantly different from that of the vectorial folding for two to eight TTAGGG repeats. Strikingly, the vectorially folded state leads to a remarkably higher accessibility to complementary C-rich strand and the telomere binding protein POT1, reflecting a less stably folded state resulting from the vectorial folding. Importantly, our study points to an inherent difference between the co-polymerizing and post-polymerized folding of telomere overhang that can impact telomere architecture and downstream processes.


Asunto(s)
G-Cuádruplex , Telómero , ADN/química , Humanos , Conformación de Ácido Nucleico , Complejo Shelterina , Telomerasa/metabolismo , Telómero/química , Telómero/genética , Proteínas de Unión a Telómeros/metabolismo
8.
Biophys J ; 122(10): 1822-1832, 2023 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-37081787

RESUMEN

Telomeres, complexes of DNA and proteins, protect ends of linear chromosomes. In humans, the two shelterin proteins TRF1 and TIN2, along with cohesin subunit SA1, were proposed to mediate telomere cohesion. Although the ability of the TRF1-TIN2 and TRF1-SA1 systems to compact telomeric DNA by DNA-DNA bridging has been reported, the function of the full ternary TRF1-TIN2-SA1 system has not been explored in detail. Here, we quantify the compaction of nanochannel-stretched DNA by the ternary system, as well as its constituents, and obtain estimates of the relative impact of its constituents and their interactions. We find that TRF1, TIN2, and SA1 work synergistically to cause a compaction of the DNA substrate, and that maximal compaction occurs if all three proteins are present. By altering the sequence with which DNA substrates are exposed to proteins, we establish that compaction by TRF1 and TIN2 can proceed through binding of TRF1 to DNA, followed by compaction as TIN2 recognizes the previously bound TRF1. We further establish that SA1 alone can also lead to a compaction, and that compaction in a combined system of all three proteins can be understood as an additive effect of TRF1-TIN2 and SA1-based compaction. Atomic force microscopy of intermolecular aggregation confirms that a combination of TRF1, TIN2, and SA1 together drive strong intermolecular aggregation as it would be required during chromosome cohesion.


Asunto(s)
Telómero , Proteína 1 de Unión a Repeticiones Teloméricas , Humanos , Proteína 1 de Unión a Repeticiones Teloméricas/química , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Telómero/metabolismo , Complejo Shelterina , ADN
9.
Nucleic Acids Res ; 49(21): 12377-12393, 2021 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-34850123

RESUMEN

Human telomeres are protected by shelterin proteins, but how telomeres maintain a dynamic structure remains elusive. Here, we report an unexpected activity of POT1 in imparting conformational dynamics of the telomere overhang, even at a monomer level. Strikingly, such POT1-induced overhang dynamics is greatly enhanced when TRF2 engages with the telomere duplex. Interestingly, TRF2, but not TRF2ΔB, recruits POT1-bound overhangs to the telomere ds/ss junction and induces a discrete stepwise movement up and down the axis of telomere duplex. The same steps are observed regardless of the length of the POT1-bound overhang, suggesting a tightly regulated conformational dynamic coordinated by TRF2 and POT1. TPP1 and TIN2 which physically connect POT1 and TRF2 act to generate a smooth movement along the axis of the telomere duplex. Our results suggest a plausible mechanism wherein telomeres maintain a dynamic structure orchestrated by shelterin.


Asunto(s)
Proteínas Recombinantes/metabolismo , Complejo Shelterina/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Humanos , Microscopía Fluorescente/métodos , Unión Proteica , Complejo Shelterina/genética , Telómero/genética , Proteínas de Unión a Telómeros/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Tripeptidil Peptidasa 1/genética , Tripeptidil Peptidasa 1/metabolismo
10.
Nucleic Acids Res ; 49(22): 13000-13018, 2021 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-34883513

RESUMEN

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2-TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein-DNA structures, and monitoring of DNA-DNA and DNA-RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA-dsDNA, dsDNA-ssDNA and dsDNA-ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.


Asunto(s)
ADN/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , ADN/química , ADN/genética , Células HeLa , Humanos , Ratones Endogámicos C57BL , Microscopía de Fuerza Atómica , Conformación de Ácido Nucleico , Unión Proteica , Complejo Shelterina/genética , Complejo Shelterina/metabolismo , Telómero/genética , Proteínas de Unión a Telómeros/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética
11.
J Biol Chem ; 297(3): 101080, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34403696

RESUMEN

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Moléculas de Adhesión Celular/fisiología , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Microscopía de Fuerza Atómica/métodos , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Unión Proteica , Isoformas de Proteínas/metabolismo , Complejo Shelterina/metabolismo , Complejo Shelterina/fisiología , Telómero/metabolismo , Proteínas de Unión a Telómeros/fisiología , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/fisiología , Proteína 2 de Unión a Repeticiones Teloméricas/fisiología
12.
Proc Natl Acad Sci U S A ; 116(37): 18435-18444, 2019 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-31451640

RESUMEN

Reactive oxygen species (ROS) play important roles in aging, inflammation, and cancer. Mitochondria are an important source of ROS; however, the spatiotemporal ROS events underlying oxidative cellular damage from dysfunctional mitochondria remain unresolved. To this end, we have developed and validated a chemoptogenetic approach that uses a mitochondrially targeted fluorogen-activating peptide (Mito-FAP) to deliver a photosensitizer MG-2I dye exclusively to this organelle. Light-mediated activation (660 nm) of the Mito-FAP-MG-2I complex led to a rapid loss of mitochondrial respiration, decreased electron transport chain complex activity, and mitochondrial fragmentation. Importantly, one round of singlet oxygen produced a persistent secondary wave of mitochondrial superoxide and hydrogen peroxide lasting for over 48 h after the initial insult. By following ROS intermediates, we were able to detect hydrogen peroxide in the nucleus through ratiometric analysis of the oxidation of nuclear cysteine residues. Despite mitochondrial DNA (mtDNA) damage and nuclear oxidative stress induced by dysfunctional mitochondria, there was a lack of gross nuclear DNA strand breaks and apoptosis. Targeted telomere analysis revealed fragile telomeres and telomere loss as well as 53BP1-positive telomere dysfunction-induced foci (TIFs), indicating that DNA double-strand breaks occurred exclusively in telomeres as a direct consequence of mitochondrial dysfunction. These telomere defects activated ataxia-telangiectasia mutated (ATM)-mediated DNA damage repair signaling. Furthermore, ATM inhibition exacerbated the Mito-FAP-induced mitochondrial dysfunction and sensitized cells to apoptotic cell death. This profound sensitivity of telomeres through hydrogen peroxide induced by dysregulated mitochondria reveals a crucial mechanism of telomere-mitochondria communication underlying the pathophysiological role of mitochondrial ROS in human diseases.


Asunto(s)
Mitocondrias/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Telómero/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , ADN Mitocondrial/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Potenciales de la Membrana , Enfermedades Mitocondriales/metabolismo , Estrés Oxidativo , Oxígeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/toxicidad , Transducción de Señal , Superóxidos/metabolismo , Superóxidos/toxicidad , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo
13.
Biochemistry ; 59(28): 2627-2639, 2020 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-32578995

RESUMEN

Telomeres are hot spots for mutagenic oxidative and methylation base damage due to their high guanine content. We used single-molecule fluorescence resonance energy transfer detection and biochemical assays to determine how different positions and types of guanine damage and mutations alter telomeric G-quadruplex structure and telomerase activity. We compared 15 modifications, including 8-oxoguanine (8oxoG), O-6-methylguanine (O6mG), and all three possible point mutations (G to A, T, and C) at the 3' three terminal guanine positions of a telomeric G-quadruplex, which is the critical access point for telomerase. We found that G-quadruplex structural instability was induced in the order C < T < A ≤ 8oxoG < O6mG, with the perturbation caused by O6mG far exceeding the perturbation caused by other base alterations. For all base modifications, the central G position was the most destabilizing among the three terminal guanines. While the structural disruption by 8oxoG and O6mG led to concomitant increases in telomerase binding and extension activity, the structural perturbation by point mutations (A, T, and C) did not, due to disrupted annealing between the telomeric overhang and the telomerase RNA template. Repositioning the same mutations away from the terminal guanines caused both G-quadruplex structural instability and elevated telomerase activity. Our findings demonstrate how a single-base modification drives structural alterations and telomere lengthening in a position-dependent manner. Furthermore, our results suggest a long-term and inheritable effect of telomeric DNA damage that can lead to telomere lengthening, which potentially contributes to oncogenesis.


Asunto(s)
G-Cuádruplex , Guanina/análisis , ARN/metabolismo , Telomerasa/metabolismo , Telómero/genética , Daño del ADN , Guanina/análogos & derivados , Guanina/metabolismo , Células HEK293 , Humanos , Mutación Puntual , Complejo Shelterina , Telómero/química , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismo
14.
Nucleic Acids Res ; 45(20): 11752-11765, 2017 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-28981887

RESUMEN

Telomeres are highly susceptible to oxidative DNA damage, which if left unrepaired can lead to dysregulation of telomere length homeostasis. Here we employed single molecule FRET, single molecule pull-down and biochemical analysis to investigate how the most common oxidative DNA lesions, 8-oxoguanine (8oxoG) and thymine glycol (Tg), regulate the structural properties of telomeric DNA and telomerase extension activity. In contrast to 8oxoG which disrupts the telomeric DNA structure, Tg exhibits substantially reduced perturbation of G-quadruplex folding. As a result, 8oxoG induces high accessibility, whereas Tg retains limited accessibility, of telomeric G-quadruplex DNA to complementary single stranded DNA and to telomere binding protein POT1. Surprisingly, the Tg lesion stimulates telomerase loading and activity to a similar degree as an 8oxoG lesion. We demonstrate that this unexpected stimulation arises from Tg-induced conformational alterations and dynamics in telomeric DNA. Despite impacting structure by different mechanisms, both 8oxoG and Tg enhance telomerase binding and extension activity to the same degree, potentially contributing to oncogenesis.


Asunto(s)
Daño del ADN , Estrés Oxidativo , Telomerasa/metabolismo , Telómero/enzimología , Secuencia de Bases , ADN/química , ADN/genética , ADN/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , G-Cuádruplex , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Células HEK293 , Humanos , Unión Proteica , Complejo Shelterina , Telómero/genética , Homeostasis del Telómero/genética , Proteínas de Unión a Telómeros/metabolismo , Timina/análogos & derivados , Timina/química , Timina/metabolismo
15.
Proc Natl Acad Sci U S A ; 113(30): 8448-53, 2016 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-27407146

RESUMEN

G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.


Asunto(s)
ARN Helicasas DEAD-box/química , ADN/química , G-Cuádruplex , RecQ Helicasas/química , Imagen Individual de Molécula/métodos , Helicasa del Síndrome de Werner/química , Secuencia de Bases , Dicroismo Circular , ARN Helicasas DEAD-box/metabolismo , ADN/genética , ADN/metabolismo , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , RecQ Helicasas/metabolismo , Especificidad por Sustrato , Telómero/genética , Telómero/metabolismo , Helicasa del Síndrome de Werner/metabolismo
16.
Nucleic Acids Res ; 44(13): 6363-76, 2016 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-27298259

RESUMEN

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA-DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.


Asunto(s)
Segregación Cromosómica/genética , Proteínas Nucleares/genética , Proteínas de Unión a Telómeros/genética , Telómero/genética , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Secuencias AT-Hook/genética , Cromátides/genética , Cromátides/ultraestructura , Proteínas de Unión al ADN/genética , Humanos , Microscopía de Fuerza Atómica , Mitosis/genética , Proteínas Nucleares/metabolismo , Telómero/ultraestructura , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo
17.
Molecules ; 22(12)2017 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-29207465

RESUMEN

Measurement of telomere length by fluorescent in situ hybridization is widely used for biomedical and epidemiological research, but there has been relatively little development of the technology in the 20 years since it was first reported. This report describes the use of dual gammaPNA (γPNA) probes that hybridize at alternating sites along a telomere and give rise to Förster resonance energy transfer (FRET) signals. Bright staining of telomeres is observed in nuclei, chromosome spreads and tissue samples. The use of FRET detection also allows for elimination of wash steps, normally required to remove unhybridized probes that would contribute to background signals. We found that these wash steps can diminish the signal intensity through the removal of bound, as well as unbound probes, so eliminating these steps not only accelerates the process but also enhances the quality of staining. Thus, γPNA FRET pairs allow for brighter and faster staining of telomeres in a wide range of research and clinical formats.


Asunto(s)
ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Hibridación Fluorescente in Situ/métodos , Telómero/metabolismo , Secuencia de Bases , Recuento de Células , Línea Celular , Colorantes Fluorescentes/química , Humanos , Estructura Molecular , Hibridación de Ácido Nucleico , Imagen Óptica/métodos , Osteosarcoma , Ácidos Nucleicos de Péptidos/metabolismo
18.
Nucleic Acids Res ; 42(21): 13096-109, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25355508

RESUMEN

Telomeres at chromosome ends are normally masked from proteins that signal and repair DNA double strand breaks (DSBs). Bulky DNA lesions can cause DSBs if they block DNA replication, unless they are bypassed by translesion (TLS) DNA polymerases. Here, we investigated roles for TLS polymerase η, (polη) in preserving telomeres following acute physical UVC exposure and chronic chemical Cr(VI) exposure, which both induce blocking lesions. We report that polη protects against cytotoxicity and replication stress caused by Cr(VI), similar to results with ultraviolet C light (UVC). Both exposures induce ataxia telangiectasia and Rad3-related (ATR) kinase and polη accumulation into nuclear foci and localization to individual telomeres, consistent with replication fork stalling at DNA lesions. Polη-deficient cells exhibited greater numbers of telomeres that co-localized with DSB response proteins after exposures. Furthermore, the genotoxic exposures induced telomere aberrations associated with failures in telomere replication that were suppressed by polη. We propose that polη's ability to bypass bulky DNA lesions at telomeres is critical for proper telomere replication following genotoxic exposures.


Asunto(s)
Daño del ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN/fisiología , Telómero/enzimología , Proteínas de la Ataxia Telangiectasia Mutada/análisis , Línea Celular , Línea Celular Transformada , Células Cultivadas , Cromo/toxicidad , Aberraciones Cromosómicas , Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/genética , Humanos , Mutágenos/toxicidad , Transducción de Señal , Telómero/efectos de los fármacos , Telómero/metabolismo , Telómero/efectos de la radiación , Rayos Ultravioleta
19.
Nucleic Acids Res ; 42(4): 2493-504, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24271387

RESUMEN

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼ 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼ 2.8-3.6 κ(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.


Asunto(s)
ADN/metabolismo , Telómero/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , ADN/química , Difusión , Unión Proteica , Estructura Terciaria de Proteína , Secuencias Repetitivas de Ácidos Nucleicos , Telómero/química , Proteína 2 de Unión a Repeticiones Teloméricas/química
20.
Nucleic Acids Res ; 41(22): 10323-33, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24038470

RESUMEN

Previous evidence indicates that telomeres resemble common fragile sites and present a challenge for DNA replication. The precise impediments to replication fork progression at telomeric TTAGGG repeats are unknown, but are proposed to include G-quadruplexes (G4) on the G-rich strand. Here we examined DNA synthesis and progression by the replicative DNA polymerase δ/proliferating cell nuclear antigen/replication factor C complex on telomeric templates that mimic the leading C-rich and lagging G-rich strands. Increased polymerase stalling occurred on the G-rich template, compared with the C-rich and nontelomeric templates. Suppression of G4 formation by substituting Li(+) for K(+) as the cation, or by using templates with 7-deaza-G residues, did not alleviate Pol δ pause sites within the G residues. Furthermore, we provide evidence that G4 folding is less stable on single-stranded circular TTAGGG templates where ends are constrained, compared with linear oligonucleotides. Artificially stabilizing G4 structures on the circular templates with the G4 ligand BRACO-19 inhibited Pol δ progression into the G-rich repeats. Similar results were obtained for yeast and human Pol δ complexes. Our data indicate that G4 formation is not required for polymerase stalling on telomeric lagging strands and suggest that an alternative mechanism, in addition to stable G4s, contributes to replication stalling at telomeres.


Asunto(s)
ADN Polimerasa III/metabolismo , ADN/biosíntesis , G-Cuádruplex , Telómero/metabolismo , ADN/química , Humanos , Secuencias Repetitivas de Ácidos Nucleicos , Moldes Genéticos
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