RESUMO
Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.
Assuntos
DNA Polimerase I , Complexo Shelterina , Proteínas de Ligação a Telômeros , Telômero , Humanos , Microscopia Crioeletrônica , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , DNA Primase/genética , Modelos Moleculares , Fosforilação , Complexo Shelterina/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres1, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand DNA synthesis ('the end-replication problem'2). Here we report a second end-replication problem that originates from the incomplete duplication of the C-rich telomeric repeat strand (C-strand) by lagging-strand DNA synthesis. This problem is resolved by fill-in synthesis mediated by polymerase α-primase bound to Ctc1-Stn1-Ten1 (CST-Polα-primase). In vitro, priming for lagging-strand DNA replication does not occur on the 3' overhang and lagging-strand synthesis stops in a zone of approximately 150 nucleotides (nt) more than 26 nt from the end of the template. Consistent with the in vitro data, lagging-end telomeres of cells lacking CST-Polα-primase lost 50-60 nt of telomeric CCCTAA repeats per population doubling. The C-strands of leading-end telomeres shortened by around 100 nt per population doubling, reflecting the generation of 3' overhangs through resection. The measured overall C-strand shortening in the absence of CST-Polα-primase fill-in is consistent with the combined effects of incomplete lagging-strand synthesis and 5' resection at the leading ends. We conclude that canonical DNA replication creates two telomere end-replication problems that require telomerase to maintain the G-rich strand and CST-Polα-primase to maintain the C-strand.
Assuntos
DNA Polimerase I , DNA Primase , Replicação do DNA , Proteínas de Ligação a Telômeros , Telômero , Humanos , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismoRESUMO
A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Sequência de Aminoácidos , Animais , Replicação do DNA , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Encurtamento do TelômeroRESUMO
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.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase I/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína BRCA1/deficiência , Linhagem Celular , DNA Primase/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Camundongos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Reparo de DNA por Recombinação , Telômero/genética , Telômero/metabolismoRESUMO
Coats plus (CP) can be caused by mutations in the CTC1 component of CST, which promotes polymerase α (polα)/primase-dependent fill-in throughout the genome and at telomeres. The cellular pathology relating to CP has not been established. We identified a homozygous POT1 S322L substitution (POT1(CP)) in two siblings with CP. POT1(CP)induced a proliferative arrest that could be bypassed by telomerase. POT1(CP)was expressed at normal levels, bound TPP1 and telomeres, and blocked ATR signaling. POT1(CP)was defective in regulating telomerase, leading to telomere elongation rather than the telomere shortening observed in other telomeropathies. POT1(CP)was also defective in the maintenance of the telomeric C strand, causing extended 3' overhangs and stochastic telomere truncations that could be healed by telomerase. Consistent with shortening of the telomeric C strand, metaphase chromosomes showed loss of telomeres synthesized by leading strand DNA synthesis. We propose that CP is caused by a defect in POT1/CST-dependent telomere fill-in. We further propose that deficiency in the fill-in step generates truncated telomeres that halt proliferation in cells lacking telomerase, whereas, in tissues expressing telomerase (e.g., bone marrow), the truncations are healed. The proposed etiology can explain why CP presents with features distinct from those associated with telomerase defects (e.g., dyskeratosis congenita).
Assuntos
Ataxia/genética , Neoplasias Encefálicas/genética , Calcinose/genética , Cistos do Sistema Nervoso Central/genética , Leucoencefalopatias/genética , Espasticidade Muscular/genética , Mutação/genética , Doenças Retinianas/genética , Convulsões/genética , Encurtamento do Telômero/genética , Proteínas de Ligação a Telômeros/genética , Telômero/genética , Telômero/patologia , Aminopeptidases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células Cultivadas , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Feminino , Humanos , Metáfase , Ligação Proteica , Serina Proteases/metabolismo , Complexo Shelterina , Transdução de Sinais , Telômero/metabolismo , Homeostase do Telômero/genéticaRESUMO
TEL2 interacts with and is essential for the stability of all phosphatidylinositol 3-kinase-related kinases (PIKKs), but its mechanism of action remains unclear. Here, we show that TEL2 is constitutively phosphorylated on conserved serines 487 and 491 by casein kinase 2 (CK2). Proteomic analyses establish that the CK2 phosphosite of TEL2 confers binding to the R2TP/prefoldin-like complex, which possesses chaperon/prefoldin activities required during protein complex assembly. The PIH1D1 subunit of the R2TP complex binds directly to the CK2 phosphosite of TEL2 in vitro and is required for the TEL2-R2TP/prefoldin-like complex interaction in vivo. Although the CK2 phosphosite mutant of TEL2 retains association with the PIKKs and HSP90 in cells, failure to interact with the R2TP/prefoldin-like complex results in instability of the PIKKs, principally mTOR and SMG1. We propose that TEL2 acts as a scaffold to coordinate the activities of R2TP/prefoldin-like and HSP90 chaperone complexes during the assembly of the PIKKs.
Assuntos
Caseína Quinase II/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Serina-Treonina Quinases TOR/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Proteínas Reguladoras de Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Sítios de Ligação/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Citoplasma/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estabilidade Enzimática , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
We reported previously that the stability of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs) depends on their interaction with Tel2, the ortholog of yeast Tel2 and Caenorhabditis elegans Clk-2. Here we provide evidence that Tel2 acts with Hsp90 in the maturation of PIKK complexes. Quantitative immunoblotting showed that the abundance of Tel2 is low compared with the PIKKs, and Tel2 preferentially bound newly synthesized ATM, ATR, mTOR, and DNA-PKcs. Tel2 complexes contained, in addition to Tti1-Tti2, the Hsp90 chaperone, and inhibition of Hsp90 interfered with the interaction of Tel2 with the PIKKs. Analysis of in vivo labeled nascent protein complexes showed that Tel2 and Hsp90 mediate the formation of the mTOR TORC1 and TORC2 complexes and the association of ATR with ATRIP. The structure of yeast Tel2, reported here, shows that Tel2 consists of HEAT-like helical repeats that assemble into two separate α-solenoids. Through mutagenesis, we identify a surface patch of conserved residues involved in binding to the Tti1-Tti2 complex in vitro. In vivo, mutation of this conserved patch affects cell growth, levels of PIKKs, and ATM/ATR-mediated checkpoint signaling, highlighting the importance of Tti1-Tti2 binding to the function of Tel2. Taken together, our data suggest that the Tel2-Tti1-Tti2 complex is a PIKK-specific cochaperone for Hsp90.
Assuntos
DNA Helicases/genética , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Células Cultivadas , DNA Helicases/química , Proteínas de Choque Térmico HSP90/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR , Proteínas de Ligação a Telômeros/genéticaRESUMO
Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(Δ/Δ) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of γH2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.
Assuntos
Antígenos Nucleares/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Espermatogênese , Testículo/patologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Antígenos Nucleares/genética , Apoptose/genética , Ciclo Celular , Células Cultivadas , Reparo do DNA/genética , DNA Recombinante/genética , Proteínas de Ligação a DNA/genética , Autoantígeno Ku , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão/genética , Recombinação Genética , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Warfarin reduces the risk of stroke in patients with atrial fibrillation, but requires a moderate-to-high time in therapeutic range (TTR). We hypothesized that point-of-care (POC) testing for prothrombin time-internationalized normalized ratio (PT-INR) could improve the TTR in patients receiving warfarin. METHODS AND RESULTS: Eight outpatient clinics that introduced POC testing for PT-INR participated in this study. We identified 148 consecutive patients who received warfarin for at least 12 months before and after the introduction of POC testing. We compared the TTR before and after the introduction of POC testing for each patient. TTR after the introduction of POC testing was significantly higher than that beforehand (51.9%±33.0% vs. 69.3%±26.3%; P<0.0001). The improvement in TTR was statistically significant in patients who had low TTR (<70%) before the introduction of POC testing. After the introduction of POC, the time spent above the target INR showed no significant change (3.7%±10.6% vs. 3.3%±6.3%, P=0.7322), while that spent below the target INR improved significantly (44.4%±34.4% vs. 27.4%±27.6%, P<0.0001). CONCLUSIONS: The introduction of POC testing was associated with an improvement in TTR, mainly through a reduction in the time spent below the target INR.
Assuntos
Instituições de Assistência Ambulatorial , Anticoagulantes/administração & dosagem , Sistemas Automatizados de Assistência Junto ao Leito , Tempo de Protrombina , Varfarina/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres1, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand DNA synthesis ("the end-replication problem"2). We report a second end-replication problem that originates from the incomplete duplication of the C-rich telomeric repeat strand by lagging-strand synthesis. This problem is solved by CST-Polymeraseα(Polα)-primase fill-in synthesis. In vitro, priming for lagging-strand DNA replication does not occur on the 3' overhang and lagging-strand synthesis stops in an ~150-nt zone more than 26 nt from the end of the template. Consistent with the in vitro data, lagging-end telomeres of cells lacking CST-Polα-primase lost ~50-60 nt of CCCTAA repeats per population doubling (PD). The C-strands of leading-end telomeres shortened by ~100 nt/PD, reflecting the generation of 3' overhangs through resection. The measured overall C-strand shortening in absence of CST-Polα-primase fill-in is consistent with the combined effects of incomplete lagging-strand synthesis and 5' resection at the leading-ends. We conclude that canonical DNA replication creates two telomere end-replication problems that require telomerase to maintain the G-strand and CST-Polα-primase to maintain the C-strand.
RESUMO
Telomere maintenance requires extension of the G-rich telomeric repeat strand by telomerase and fill-in synthesis of the C-rich strand by Polα/Primase. Telomeric Polα/Primase is bound to Ctc1-Stn1-Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/Primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Phosphorylation of POT1 is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/Primase in an inactive auto-inhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/Primase into an active state that completes telomere replication through fill-in synthesis.
RESUMO
With the aging of the population, the number of people taking antithrombotic drugs is increasing. Few reports have described the clinical presentation, treatment, and outcomes of nontraumatic subarachnoid hemorrhage (SAH) in patients with preceding antithrombotic therapy. This study included 459 patients with nontraumatic SAH who had been treated between April 2009 and May 2021. Overall, 39 of the 459 patients with aneurysmal SAH were on antithrombotic therapy before ictus (8.5%). Therefore, we classified patients into two groups: Group A (n = 39), patients with preceding antithrombotic therapy and Group B (n = 420), patients without preceding antithrombotic therapy. Hunt and Kosnik (H&K) grade on admission was significantly higher in Group A than in Group B (p = 0.02). Patients in Group A more frequently received endovascular treatment. The rate of endovascular therapy for symptomatic vasospasm after SAH was significantly lower in Group A (2.6%) than in Group B (15.5%; p = 0.03). The outcomes at 3 months after onset were significantly poorer in Group A patients than in Group B patients (p = 0.03). Patients with preceding antithrombotic drugs tended to be at greater risk of unfavorable outcomes, but this difference was not significant in the univariate analysis. In the multivariate analysis, patient age, H&K grade ≥4, and subdural hematoma remained as risk factors for poor outcomes; however, preceding use of antithrombotic drugs was not a significant risk factor.
Assuntos
Hemorragia Subaracnóidea , Humanos , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/terapia , Fibrinolíticos/efeitos adversos , Prognóstico , Hematoma Subdural , Fatores de Risco , Resultado do TratamentoRESUMO
We compared the rate of selective shunt and pattern of monitoring change between single and dual monitoring in patients undergoing carotid endarterectomy (CEA). A total of 121 patients underwent 128 consecutive CEA procedures. Excluding five procedures using internal shunts in a premeditated manner, we classified patients according to the monitoring: Group A (n = 72), patients with single somatosensory evoked potential (SSEP) monitoring; and Group B (n = 51), patients with dual SSEP and motor evoked potential (MEP). Among the 123 CEAs, an internal shunt was inserted in 12 procedures (9.8%) due to significant changes in monitoring (Group A 5.6%, Group B 15.7%, p = 0.07). The rate of shunt use was significantly higher in patients with the absence of contralateral proximal anterior cerebral artery (A1) on magnetic resonance angiography (MRA) than in patients with other types of MRA (p <0.001). Significant monitor changes were seen in 16 (12.5%) in both groups. In four of nine patients in Group B, SSEP and MEP changes were synchronized, and in the remaining five patients, a time lag was evident between SSEP and MEP changes. In conclusion, the rate of internal shunt use tended to be more frequent in patients with dual monitoring than in patients with single SSEP monitoring, but the difference was not significant. Contralateral A1 absence may predict the need for a shunt and care should be taken to monitor changes throughout the entire CEA procedure. Use of dual monitoring can capture ischemic changes due to the complementary relationship, and may reduce the rate of false-negative monitor changes during CEA.
Assuntos
Artéria Carótida Interna/cirurgia , Estenose das Carótidas/cirurgia , Endarterectomia das Carótidas/efeitos adversos , Potencial Evocado Motor , Potenciais Somatossensoriais Evocados , Monitorização Neurofisiológica Intraoperatória/métodos , Idoso , Derivação Arteriovenosa Cirúrgica , Isquemia Encefálica/diagnóstico por imagem , Artéria Carótida Interna/patologia , Artérias Cerebrais/diagnóstico por imagem , Endarterectomia das Carótidas/métodos , Feminino , Humanos , Complicações Intraoperatórias/diagnóstico por imagem , Japão , Angiografia por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Although carotid endarterectomy (CEA) is an established procedure, technical modifications are required when anatomical features are unusual. The present study aimed to determine the characteristics of diagnostic features, surgical management, and outcomes of patients with a twisted carotid bifurcation (TCB). We assessed 108 consecutive patients by cervical carotid echography (CCE) and black-blood magnetic resonance imaging (BB-MRI) before they underwent 115 CEA procedures. We classified carotid bifurcation (CB) anatomy based on anteroposterior findings of the internal carotid artery (ICA) and external carotid artery (ECA) determined by cerebral or three-dimensional computed tomographic angiography as follows. The ICA and ECA ran laterally and medially, respectively, in Type 1, overlapped in Type 2, and the ICA and ECA ran medially and laterally, respectively, in Type 3. We also classified the patients according to whether or not they had a TCB and compared their diagnostic findings, clinical characteristics, and surgical outcomes. The numbers of patients with Types 1, 2, and 3 were 74 (64.4%), 32 (27.8%), and 9 (7.8%), respectively, and 13 (11.3%) with a TCB included four patients with Type 2 and all nine patients with Type 3. The appearance of Type 3 differed from that of the other two types on CCE and BB-MR images. After correcting the anatomical location of a TCB, surgical duration and adverse event rates did not significantly differ between patients with and without a TCB. Patients with a TCB could safely undergo CEA after correcting the ICA to the normal position.
Assuntos
Artéria Carótida Externa/patologia , Artéria Carótida Interna/patologia , Estenose das Carótidas/diagnóstico , Estenose das Carótidas/cirurgia , Endarterectomia das Carótidas , Idoso , Artéria Carótida Externa/diagnóstico por imagem , Artéria Carótida Interna/diagnóstico por imagem , Estenose das Carótidas/complicações , Angiografia por Tomografia Computadorizada , Feminino , Humanos , Angiografia por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , UltrassonografiaRESUMO
Local wet etching technique was proposed to fabricate high-performance aspherical mirrors. In this process, only the limited area facing to the small nozzle is removed by etching on objective surface. The desired objective shape is deterministically fabricated by performing the numerically controlled scanning of the nozzle head. Using the technique, a plano-elliptical mirror to focus the neutron beam was successfully fabricated with the figure accuracy of less than 0.5 microm and the focusing gain of 6. The strong and thin focused neutron beam is expected to be a useful tool for the analyses of various material properties.
Assuntos
Lentes , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Nêutrons , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Propriedades de SuperfícieRESUMO
We report cytologic and genetic data indicating that telomere dysfunction induces a DNA damage response in mammalian cells. Dysfunctional, uncapped telomeres, created through inhibition of TRF2, became associated with DNA damage response factors, such as 53BP1, gamma-H2AX, Rad17, ATM, and Mre11. We refer to the domain of telomere-associated DNA damage factors as a Telomere Dysfunction-Induced Focus (TIF). The accumulation of 53BP1 on uncapped telomeres was reduced in the presence of the PI3 kinase inhibitors caffeine and wortmannin, which affect ATM, ATR, and DNA-PK. By contrast, Mre11 TIFs were resistant to caffeine, consistent with previous findings on the Mre11 response to ionizing radiation. A-T cells had a diminished 53BP1 TIF response, indicating that the ATM kinase is a major transducer of this pathway. However, in the absence of ATM, TRF2 inhibition still induced TIFs and senescence, pointing to a second ATM-independent pathway. We conclude that the cellular response to telomere dysfunction is governed by proteins that also control the DNA damage response. TIFs represent a new tool for evaluating telomere status in normal and malignant cells suspected of harboring dysfunctional telomeres. Furthermore, induction of TIFs through TRF2 inhibition provides an opportunity to study the DNA damage response within the context of well-defined, physically marked lesions.
Assuntos
Dano ao DNA/genética , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Proteínas de Transporte/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Imunofluorescência , Células HeLa , Humanos , Immunoblotting , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/antagonistas & inibidores , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
The human telomeric DNA binding factor TRF1 (hTRF1) and its interacting proteins TIN2, tankyrase 1 and 2, and PINX1 have been implicated in the regulation of telomerase-dependent telomere length maintenance. Here we show that targeted deletion of exon 1 of the mouse gene encoding Trf1 causes early (day 5 to 6 postcoitus) embryonic lethality. The absence of telomerase did not alter the Terf1(ex1Delta/ex1Delta) lethality, indicating that the phenotype was not due to inappropriate telomere elongation by telomerase. Terf1(ex1Delta/ex1Delta) blastocysts had a severe growth defect of the inner cell mass that was accompanied by apoptosis. However, no evidence was found for telomere uncapping causing this cell death; chromosome spreads of Terf1(ex1Delta/ex1Delta) blastocysts did not reveal chromosome end-to-end fusions, and p53 deficiency only briefly delayed Terf1(ex1Delta/ex1Delta) lethality. These data suggest that murine Trf1 has an essential function that is independent of telomere length regulation.
Assuntos
Telômero/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Animais , Blastocisto/patologia , Divisão Celular , Células Cultivadas , Feminino , Morte Fetal/genética , Marcação de Genes , Camundongos , Camundongos Mutantes , Gravidez , Deleção de Sequência , Telomerase/deficiência , Telomerase/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
A growing number of DNA polymerases have been identified, although their physiological function and relation to human disease remain mostly unknown. DNA polymerase lambda (Pol lambda; also known as Pol beta2) has recently been identified as a member of the X family of DNA polymerases and shares 32% amino acid sequence identity with DNA Pol beta within the polymerase domain. With the use of homologous recombination, we generated Pol lambda(-/-) mice. Pol lambda(-/-) mice develop hydrocephalus with marked dilation of the lateral ventricles and exhibit a high rate of mortality after birth, although embryonic development appears normal. Pol lambda(-/-) mice also show situs inversus totalis and chronic suppurative sinusitis. The surviving male, but not female, Pol lambda(-/-) mice are sterile as a result of spermatozoal immobility. Microinjection of sperm from male Pol lambda(-/-) mice into oocytes gives rise to normal offspring, suggesting that the meiotic process is not impaired. Ultrastructural analysis reveals that inner dynein arms of cilia from both the ependymal cell layer and respiratory epithelium are defective, which may underlie the pathogenesis of hydrocephalus, situs inversus totalis, chronic sinusitis, and male infertility. Sensitivity of Pol lambda(-/-) cells to various kinds of DNA damage is indistinguishable from that of Pol lambda(+/+) cells. Collectively, Pol lambda(-/-) mice may provide a useful model for clarifying the pathogenesis of immotile cilia syndrome.
Assuntos
Transtornos da Motilidade Ciliar/etiologia , DNA Polimerase beta/deficiência , DNA Polimerase beta/genética , Hidrocefalia/enzimologia , Hidrocefalia/genética , Infertilidade Masculina/enzimologia , Infertilidade Masculina/genética , Sinusite/enzimologia , Sinusite/genética , Situs Inversus/enzimologia , Situs Inversus/genética , Animais , Doença Crônica , Transtornos da Motilidade Ciliar/enzimologia , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , DNA Polimerase beta/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Hidrocefalia/patologia , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , Gravidez , Sinusite/patologia , Situs Inversus/patologia , Injeções de Esperma IntracitoplásmicasRESUMO
The ventricular septum receives its blood supply from the septal perforators of the left anterior descending (LAD) coronary artery and the right coronary artery. However, when the LAD artery extends to the inferior wall, beyond the apex (so-called wrapped LAD), the ventricular septum near the apex receives blood supply only from the LAD artery. As a consequence, ventricular septal rupture (VSR) would seem more likely in myocardial infarction with occlusion of this type of LAD artery. To test this hypothesis, we compared electrocardiographic findings in 21 patients who had anterior acute myocardial infarction that was complicated by VSR with those in 275 patients who had acute myocardial infarction that was not complicated by VSR. We observed ST-segment elevation in all inferior leads (II, III, and aVF) in addition to anterior leads in 42.9% of patients (9 of 21) who had VSR but in only 3.6% of those (10 of 275) who did not have VSR. Abnormal Q waves appeared in all 3 inferior leads in 44.4% of patients (8 of 18) who had VSR but in only 4.0% of those (10 of 250) who did not have VSR. Thus, the incidence of ST-segment elevation and abnormal Q waves in the inferior leads was significantly (p <0.001) greater in the VSR group. In addition, multivariate analysis of patient characteristics, including advanced age, female gender, and coronary morphology, showed VSR to be significantly correlated with ST-segment elevation (odds ratio 16.93, 95% confidence interval 4.13 to 69.30) and abnormal Q waves (odds ratio 13.64, 95% confidence interval 3.16 to 58.79) in the 3 inferior leads. In conclusion, these electrocardiographic findings can be useful predictors of complication by VSR.