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1.
Genetics ; 219(2)2021 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-34849883

RESUMO

Large-scale expansion of (GAA)n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich's ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system. The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions. This screen, however, also produced two unexpected hits: members of the CST complex, CDC13 and TEN1 genes, which are required for telomere maintenance. To understand how the CST complex could affect intra-chromosomal GAA repeats, we studied the well-characterized temperature-sensitive cdc13-1 mutation and its effects on GAA repeat instability in yeast. We found that in-line with the screen results, this mutation leads to ∼10-fold increase in the rate of large-scale expansions of the (GAA)100 repeat at semi-permissive temperature. Unexpectedly, the hyper-expansion phenotype of the cdc13-1 mutant largely depends on activation of the G2/M checkpoint, as deletions of individual genes RAD9, MEC1, RAD53, and EXO1 belonging to this pathway rescued the increased GAA expansions. Furthermore, the hyper-expansion phenotype of the cdc13-1 mutant depended on the subunit of DNA polymerase δ, Pol32. We hypothesize, therefore, that increased repeat expansions in the cdc13-1 mutant happen during post-replicative repair of nicks or small gaps within repetitive tracts during the G2 phase of the cell cycle upon activation of the G2/M checkpoint.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Expansão das Repetições de Trinucleotídeos , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
2.
Ophthalmic Genet ; 41(6): 656-658, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32940098

RESUMO

INTRODUCTION: Recently, You, Hoover-Fong, and colleagues described a disease caused by a deficiency of the telomere maintenance 2 gene (TELO2) function. The clinical spectrum includes early-onset global delay, dysmorphic facial features, auditory disorder, and reduced vision. MATERIALS AND METHODS: We report two siblings, diagnosed with You-Hoover-Fong syndrome at the age of 28 and 14 months. Both were genetically studied to find the cause of their developmental delay and microcephaly. RESULTS: The identical compound heterozygous missense mutations in the TELO2gene were found in each. Ophthalmologically, both siblings were diagnosed with progressive congenital bilateral nuclear-lamellar cataracts. CONCLUSIONS: We report nuclear-lamellar cataracts in two siblings diagnosed with You-Hoover-Fong syndrome.


Assuntos
Catarata/patologia , Deficiências do Desenvolvimento/patologia , Deficiência Intelectual/patologia , Microcefalia/patologia , Mutação de Sentido Incorreto , Homeostase do Telômero , Proteínas de Ligação a Telômeros/deficiência , Catarata/etiologia , Pré-Escolar , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/genética , Feminino , Humanos , Lactente , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Masculino , Microcefalia/complicações , Microcefalia/genética , Irmãos , Proteínas de Ligação a Telômeros/metabolismo
3.
Cell Rep ; 29(11): 3708-3725.e5, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31825846

RESUMO

Telomeres use shelterin to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), repressing ataxia-telangiectasia, mutated (ATM) and ATM and Rad3-related (ATR) dependent DNA damage checkpoint responses. The MRE11 nuclease is thought to be essential for the resection of the 5' C-strand to generate the microhomologies necessary for alternative non-homologous end joining (A-NHEJ) repair. In the present study, we uncover DNA damage signaling and repair pathways engaged by components of the replisome complex to repair dysfunctional telomeres. In cells lacking MRN, single-stranded telomeric overhangs devoid of POT1-TPP1 do not recruit replication protein A (RPA), ATR-interacting protein (ATRIP), and RAD 51. Rather, components of the replisome complex, including Claspin, Proliferating cell nuclear antigen (PCNA), and Downstream neighbor of SON (DONSON), initiate DNA-PKcs-mediated p-CHK1 activation and A-NHEJ repair. In addition, Claspin directly interacts with TRF2 and recruits EXO1 to newly replicated telomeres to promote 5' end resection. Our data indicate that MRN is dispensable for the repair of dysfunctional telomeres lacking POT1-TPP1 and highlight the contributions of the replisome in telomere repair.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Telômero/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aminopeptidases/deficiência , Aminopeptidases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Quinase 1 do Ponto de Checagem/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/deficiência , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Exodesoxirribonucleases/metabolismo , Células HEK293 , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , Complexos Multienzimáticos/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Serina Proteases/deficiência , Serina Proteases/metabolismo , Complexo Shelterina , Telômero/genética , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
4.
Nature ; 574(7779): 571-574, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645724

RESUMO

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-POLα complex2. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Instabilidade Genômica , Conformação de Ácido Nucleico , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
5.
Mol Biol Cell ; 30(14): 1664-1675, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31091167

RESUMO

The cardiomyocyte cytoskeleton, including the sarcomeric contractile apparatus, forms a cohesive network with cellular adhesions at the plasma membrane and nuclear--cytoskeletal linkages (LINC complexes) at the nuclear envelope. Human cardiomyopathies are genetically linked to the LINC complex and A-type lamins, but a full understanding of disease etiology in these patients is lacking. Here we show that SUN2-null mice display cardiac hypertrophy coincident with enhanced AKT/MAPK signaling, as has been described previously for mice lacking A-type lamins. Surprisingly, in contrast to lamin A/C-null mice, SUN2-null mice fail to show coincident fibrosis or upregulation of pathological hypertrophy markers. Thus, cardiac hypertrophy is uncoupled from profibrotic signaling in this mouse model, which we tie to a requirement for the LINC complex in productive TGFß signaling. In the absence of SUN2, we detect elevated levels of the integral inner nuclear membrane protein MAN1, an established negative regulator of TGFß signaling, at the nuclear envelope. We suggest that A-type lamins and SUN2 play antagonistic roles in the modulation of profibrotic signaling through opposite effects on MAN1 levels at the nuclear lamina, suggesting a new perspective on disease etiology.


Assuntos
Cardiomegalia/metabolismo , Cardiomegalia/patologia , Deleção de Genes , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Adesão Celular , Forma do Núcleo Celular , Proteínas de Ligação a DNA/metabolismo , Fibrose , Integrinas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Membrana Nuclear/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Proteínas de Ligação a Telômeros/deficiência , Fator de Crescimento Transformador beta/metabolismo
6.
Nucleic Acids Res ; 45(8): 4281-4293, 2017 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-28334750

RESUMO

To prevent progressive telomere shortening as a result of conventional DNA replication, new telomeric DNA must be added onto the chromosome end. The de novo DNA synthesis involves elongation of the G-rich strand of the telomere by telomerase. In human cells, the CST complex (CTC1-STN1-TEN1) also functions in telomere replication. CST first aids in duplication of the telomeric dsDNA. Then after telomerase has extended the G-rich strand, CST facilitates fill-in synthesis of the complementary C-strand. Here, we analyze telomere structure after disruption of human CTC1 and demonstrate that functional CST is essential for telomere length maintenance due to its role in mediating C-strand fill-in. Removal of CTC1 results in elongation of the 3΄ overhang on the G-rich strand. This leads to accumulation of RPA and telomeric DNA damage signaling. G-overhang length increases with time after CTC1 disruption and at early times net G-strand growth is apparent, indicating telomerase-mediated G-strand extension. In contrast, C-strand length decreases continuously, indicating a deficiency in C-strand fill-in synthesis. The lack of C-strand maintenance leads to gradual shortening of the telomeric dsDNA, similar to that observed in cells lacking telomerase. Thus, telomerase-mediated G-strand extension and CST-mediated C-strand fill-in are equally important for telomere length maintenance.


Assuntos
DNA/química , Telomerase/genética , Homeostase do Telômero , Proteínas de Ligação a Telômeros/genética , Telômero/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Replicação do DNA , Deleção de Genes , Regulação da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Telomerase/metabolismo , Telômero/ultraestrutura , Encurtamento do Telômero , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/metabolismo
7.
Dev Biol ; 418(2): 258-67, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27565025

RESUMO

Plexins (Plexs) comprise a large family of cell surface receptors for semaphorins (Semas) that function as evolutionarily conserved guidance molecules. GTPase activating protein (GAP) activity for Ras family small GTPases has been implicated in plexin signaling cascades through its RasGAP domain. However, little is known about how Ras family GTPases are controlled in vivo by plexin signaling. Here, we found that Drosophila Rap1, a member of the Ras family of GTPases, plays an important role controlling intersegmental nerve b motor axon guidance during neural development. Gain-of-function studies using dominant-negative and constitutively active forms of Rap1 indicate that Rap1 contributes to axonal growth and guidance. Genetic interaction analyses demonstrate that the Sema-1a/PlexA-mediated repulsive guidance function is regulated positively by Rap1. Furthermore, neuronal expression of mutant PlexA robustly restored defasciculation defects in PlexA null mutants when the catalytic arginine fingers of the PlexA RasGAP domain critical for GAP activity were disrupted. However, deleting the RasGAP domain abolished the ability of PlexA to rescue the PlexA guidance phenotypes. These findings suggest that PlexA-mediated motor axon guidance is dependent on the presence of the PlexA RasGAP domain, but not on its GAP activity toward Ras family small GTPases.


Assuntos
Orientação de Axônios/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores de Superfície Celular/fisiologia , Proteínas de Ligação a Telômeros/fisiologia , Proteínas Ativadoras de ras GTPase/fisiologia , Animais , Animais Geneticamente Modificados , Orientação de Axônios/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insetos , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/genética , Neurônios Motores/fisiologia , Mutagênese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Complexo Shelterina , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/genética , Regulação para Cima , Proteínas Ativadoras de ras GTPase/deficiência , Proteínas Ativadoras de ras GTPase/genética
8.
J Genet Genomics ; 43(7): 451-65, 2016 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-27477026

RESUMO

In the budding yeast Saccharomyces cerevisiae, heterochromatin structure is found at three chromosome regions, which are homothallic mating-type loci, rDNA regions and telomeres. To address how telomere heterochromatin is assembled under physiological conditions, we employed a de novo telomere addition system, and analyzed the dynamic chromatin changes of the TRP1 reporter gene during telomere elongation. We found that integrating a 255-bp, but not an 81-bp telomeric sequence near the TRP1 promoter could trigger Sir2 recruitment, active chromatin mark(s)' removal, chromatin compaction and TRP1 gene silencing, indicating that the length of the telomeric sequence inserted in the internal region of a chromosome is critical for determining the chromatin state at the proximal region. Interestingly, Rif1 but not Rif2 or yKu is indispensable for the formation of intra-chromosomal silent chromatin initiated by telomeric sequence. When an internal short telomeric sequence (e.g., 81 bp) gets exposed to become a de novo telomere, the herterochromatin features, such as Sir recruitment, active chromatin mark(s)' removal and chromatin compaction, are detected within a few hours before the de novo telomere reaches a stable length. Our results recapitulate the molecular dynamics and reveal a coherent picture of telomere heterochromatin formation.


Assuntos
Heterocromatina/genética , Saccharomyces cerevisiae/genética , Telômero/genética , Aldose-Cetose Isomerases/deficiência , Aldose-Cetose Isomerases/genética , Sequência de Bases , Inativação Gênica , Loci Gênicos/genética , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/genética
10.
Cell Rep ; 9(4): 1273-80, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25453752

RESUMO

The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ) at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN)-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3' telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres.


Assuntos
Sequência Conservada , Endonucleases/metabolismo , Técnicas de Inativação de Genes , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/genética , Telômero/metabolismo , Transativadores/metabolismo , Animais , Proliferação de Células , Cromatina/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Complexo Shelterina , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/metabolismo , Transcrição Gênica
11.
Mol Cell Biol ; 34(7): 1349-62, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24469404

RESUMO

The shelterin protein TIN2 is required for the telomeric accumulation of TPP1/POT1 heterodimers and for the protection of telomeres by the POT1 proteins (POT1a and POT1b in the mouse). TIN2 also binds to TRF1 and TRF2, improving the telomeric localization of TRF2 and its function. Here, we ask whether TIN2 needs to interact with both TRF1 and TRF2 to mediate the telomere protection afforded by TRF2 and POT1a/b. Using a TIN2 allele deficient in TRF1 binding (TIN2-L247E), we demonstrate that TRF1 is required for optimal recruitment of TIN2 to telomeres and document phenotypes associated with the TIN2-L247E allele that are explained by insufficient TIN2 loading onto telomeres. To bypass the requirement for TRF1-dependent recruitment, we fused TIN2-L247E to the TRF2-interacting (RCT) domain of Rap1. The RCT-TIN2-L247E fusion showed improved telomeric localization and was fully functional in terms of chromosome end protection by TRF2, TPP1/POT1a, and TPP1/POT1b. These data indicate that when sufficient TIN2 is loaded onto telomeres, its interaction with TRF1 is not required to mediate the function of TRF2 and the TPP1/POT1 heterodimers. We therefore conclude that shelterin can protect chromosome ends as a TRF2-tethered TIN2/TPP1/POT1 complex that lacks a physical connection to TRF1.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Animais , Células Cultivadas , Camundongos , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Complexo Shelterina , Telômero/genética , Homeostase do Telômero/genética , Homeostase do Telômero/fisiologia , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação a Repetições Teloméricas/química , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genética
12.
J Clin Invest ; 124(1): 353-66, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24316971

RESUMO

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Mutação , Proteínas de Ligação a Telômeros/genética , Animais , Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Instabilidade Cromossômica , Aberrações Cromossômicas , Ativação Enzimática , Pontos de Checagem da Fase G2 do Ciclo Celular , Genes Letais , Transplante de Células-Tronco Hematopoéticas , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pancitopenia/genética , Encurtamento do Telômero , Proteínas de Ligação a Telômeros/deficiência
13.
EMBO J ; 31(24): 4576-86, 2012 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-23188080

RESUMO

Telomeres protect the natural ends of chromosomes from being repaired as deleterious DNA breaks. In fission yeast, absence of Taz1 (homologue of human TRF1 and TRF2) renders telomeres vulnerable to DNA repair. During the G1 phase, when non-homologous end joining (NHEJ) is upregulated, taz1Δ cells undergo telomere fusions with consequent loss of viability. Here, we show that disruption of the fission yeast MRN (Rad23(MRE11)-Rad50-Nbs1) complex prevents NHEJ at telomeres and, as a result, rescues taz1Δ lethality in G1. Neither Tel1(ATM) activation nor 5'-end resection was required for telomere fusion. Nuclease activity of Rad32(MRE11) was also dispensable for NHEJ. Mutants unable to coordinate metal ions required for nuclease activity were proficient in NHEJ repair. In contrast, Rad32(MRE11) mutations that affect binding and/or positioning of DNA ends leaving the nuclease function largely unaffected also impaired NHEJ at telomeres and restored the viability of taz1Δ in G1. Consistently, MRN structural integrity but not nuclease function is also required for NHEJ of independent DNA ends in a novel split-molecule plasmid assay. Thus, MRN acts to tether unlinked DNA ends, allowing for efficient NHEJ.


Assuntos
Reparo do DNA por Junção de Extremidades/fisiologia , Fase G1/fisiologia , Complexos Multiproteicos/metabolismo , Telômero/metabolismo , Sobrevivência Celular/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Primers do DNA/genética , Eletroforese em Gel de Campo Pulsado , Exodesoxirribonucleases/metabolismo , Reação em Cadeia da Polimerase , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ligação a Telômeros/deficiência
14.
Proc Natl Acad Sci U S A ; 109(20): 7805-10, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22547822

RESUMO

Canonical telomere repeats at chromosome termini can be maintained by a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). Human cancers that survive via ALT can exhibit long and heterogeneous telomeres, although many telomerase-negative tumors possess telomeres of normal length. Here, we report that Caenorhabditis elegans telomerase mutants that survived via ALT possessed either long or normal telomere lengths. Most ALT strains displayed end-to-end chromosome fusions, suggesting that critical telomere shortening occurred before or concomitant with ALT. ALT required the 9-1-1 DNA damage response complex and its clamp loader, HPR-17. Deficiency for the POT-2 telomere binding protein promoted ALT in telomerase mutants, overcame the requirement for the 9-1-1 complex in ALT, and promoted ALT with normal telomere lengths. We propose that telomerase-deficient human tumors with normal telomere lengths could represent a mode of ALT that is facilitated by telomere capping protein dysfunction.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Homeostase do Telômero/fisiologia , Proteínas de Ligação a Telômeros/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Indóis , Mutação/genética , Polimorfismo de Fragmento de Restrição , Telomerase/genética , Proteínas de Ligação a Telômeros/deficiência
15.
EMBO J ; 31(8): 2024-33, 2012 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-22425786

RESUMO

To counteract replication-dependent telomere shortening most eukaryotic cells rely on the telomerase pathway, which is crucial for the maintenance of proliferative potential of germ and stem cell populations of multicellular organisms. Likewise, cancer cells usually engage the telomerase pathway for telomere maintenance to gain immortality. However, in ∼10% of human cancers telomeres are maintained through telomerase-independent alternative lengthening of telomeres (ALT) pathways. Here, we describe the generation and characterization of C. elegans survivors in a strain lacking the catalytic subunit of telomerase and the nematode telomere-binding protein CeOB2. These clonal strains, some of which have been propagated for >180 generations, represent the first example of a multicellular organism with canonical telomeres that can survive without a functional telomerase pathway. The animals display the heterogeneous telomere length characteristic for ALT cells, contain single-stranded C-circles, a transcription profile pointing towards an adaptation to chronic stress and are therefore a unique and valuable tool to decipher the ALT mechanism.


Assuntos
Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/crescimento & desenvolvimento , Telomerase/deficiência , Proteínas de Ligação a Telômeros/deficiência , Telômero/metabolismo , Animais , Caenorhabditis elegans/genética , Análise de Sobrevida
16.
Mol Cell ; 39(4): 606-17, 2010 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-20619712

RESUMO

Mammalian telomeres contain a single-stranded 3' overhang that is thought to mediate telomere protection. Here we identify the TRF2-interacting factor Apollo as a nuclease that contributes to the generation/maintenance of this overhang. The function of mouse Apollo was determined using Cre-mediated gene deletion, complementation with Apollo mutants, and the TRF2-F120A mutant that cannot bind Apollo. Cells lacking Apollo activated the ATM kinase at their telomeres in S phase and showed leading-end telomere fusions. These telomere dysfunction phenotypes were accompanied by a reduction in the telomeric overhang signal. The telomeric functions of Apollo required its TRF2-interaction and nuclease motifs. Thus, TRF2 recruits the Apollo nuclease to process telomere ends synthesized by leading-strand DNA synthesis, thereby creating a terminal structure that avoids ATM activation and resists end-joining. These data establish that the telomeric overhang is required for the protection of telomeres from the DNA damage response.


Assuntos
Replicação do DNA , Fibroblastos/enzimologia , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Motivos de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Exodesoxirribonucleases , Fase G2 , Fusão Gênica , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Conformação de Ácido Nucleico , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Fase S , Telômero/química , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismo
17.
Eur J Cancer Prev ; 19(5): 345-51, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20517159

RESUMO

Telomeres are the end structures of linear chromosomes in eukaryotic cells. The integrity of a telomere is essential for the overall stability of the chromosome. The human protection of telomeres 1 (hPOT1) protein, a single-stranded telomeric DNA binding protein, plays an important role in telomere protection and telomere length regulation. Here, we show that the loss of hPOT1 by RNA interference in BGC823 (poorly differentiated human gastric adenocarcinoma) cells leads to an increase in multinucleated giant cells, a decrease in cell proliferation and colony formation, induction of senescence and apoptosis, shortened telomere length, upregulation of the TRF1 gene and downregulation of the TRF2, tankyrase1 and hTERT genes. These results suggest that the loss of hPOT1 results in a decrease in the viability of BGC823 cells; hPOT1 regulates telomere length positively and has an influence on the expression of other telomere-associated genes in the cells.


Assuntos
Adenocarcinoma/genética , Neoplasias Gástricas/genética , Proteínas de Ligação a Telômeros/deficiência , Telômero/genética , Telômero/ultraestrutura , Adenocarcinoma/metabolismo , Sobrevivência Celular , Expressão Gênica , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Complexo Shelterina , Neoplasias Gástricas/metabolismo , Tanquirases/genética , Telomerase/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Células Tumorais Cultivadas
18.
Mol Cell Biol ; 30(12): 2971-82, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20404094

RESUMO

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres--TPP1 and the protein that tethers TPP1 to the complex, TIN2--results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.


Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/enzimologia , Imunoprecipitação da Cromatina , Dano ao DNA , Células HeLa , Humanos , Ligação Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Complexo Shelterina , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/deficiência
19.
Neuron ; 64(2): 173-87, 2009 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-19874786

RESUMO

Nuclear movement is critical during neurogenesis and neuronal migration, which are fundamental for mammalian brain development. Although dynein, Lis1, and other cytoplasmic proteins are known for their roles in connecting microtubules to the nucleus during interkinetic nuclear migration (INM) and nucleokinesis, the factors connecting dynein/Lis1 to the nuclear envelope (NE) remain to be determined. We report here that the SUN-domain proteins SUN1 and SUN2 and the KASH-domain proteins Syne-1/Nesprin-1 and Syne-2/Nesprin-2 play critical roles in neurogenesis and neuronal migration in mice. We show that SUN1 and SUN2 redundantly form complexes with Syne-2 to mediate the centrosome-nucleus coupling during both INM and radial neuronal migration in the cerebral cortex. Syne-2 is connected to the centrosome through interactions with both dynein/dynactin and kinesin complexes. Syne-2 mutants also display severe defects in learning and memory. These results fill an important gap in our understanding of the mechanism of nuclear movement during brain development.


Assuntos
Centrossomo/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neurônios/citologia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Comportamento Animal/fisiologia , Encéfalo/citologia , Bromodesoxiuridina/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Proteínas do Citoesqueleto , Dineínas/metabolismo , Eletroporação/métodos , Comportamento Exploratório/fisiologia , Feminino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas do Tecido Nervoso/deficiência , Neurogênese/genética , Neurônios/ultraestrutura , Proteínas Nucleares/deficiência , Gravidez , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína/genética , Transporte Proteico/fisiologia , Proteínas de Ligação a Telômeros/deficiência
20.
Mol Cell ; 33(5): 559-69, 2009 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-19285940

RESUMO

Genome stability depends upon the RecQ helicases, which are conserved from bacteria to man, but little is known about how their myriad activities are regulated. Fission yeast lacking the telomere protein Taz1 (mammalian TRF1/TRF2 ortholog) lose many hallmarks of telomeres, including accurate replication and local protection from DNA repair reactions. Here we show that the RecQ homolog, Rqh1, is sumoylated. Surprisingly, Rqh1 acts on taz1Delta telomeres in a deleterious way, promoting telomere breakage and entanglement. Mutation of Rqh1 sumoylation sites rescues taz1Delta cells from these hazards without dramatically affecting nontelomeric Rqh1 functions. The prominence of Rqh1 in the etiology of several different telomere defects supports the idea that they originate from a common underlying lesion--aberrant processing of the stalled telomeric replication forks that accumulate in the absence of Taz1. Our work underscores the principle that RecQ helicases are "double-edged swords" whose activity, while necessary for maintaining genome-wide stability, must be vigilantly controlled.


Assuntos
DNA Helicases/metabolismo , Regulação Fúngica da Expressão Gênica , Processamento de Proteína Pós-Traducional , RecQ Helicases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Telômero/metabolismo , Alelos , Temperatura Baixa , DNA Helicases/genética , Replicação do DNA , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Instabilidade Genômica , Genótipo , Mutação , Fenótipo , RecQ Helicases/genética , Recombinação Genética , Origem de Replicação , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/efeitos da radiação , Estresse Fisiológico/genética , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/genética , Fatores de Tempo
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