RESUMO
Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that interacts with the replisome to aid in stalled fork rescue. We previously found that CST promotes telomere replication to maintain genomic integrity via G-quadruplex (G4) resolution. However, the detailed mechanism by which CST resolves G4s in vivo and whether additional factors are involved remains unclear. Here, we identify RECQ4 as a novel CST-interacting partner and show that RECQ4 can unwind G4 structures in vitro using a FRET assay. Moreover, G4s accumulate at the telomere after RECQ4 depletion, resulting in telomere dysfunction, including the formation of MTSs, SFEs, and TIFs, suggesting that RECQ4 is crucial for telomere integrity. Furthermore, CST is also required for RECQ4 telomere or chromatin localization in response to G4 stabilizers. RECQ4 is involved in preserving genomic stability by CST and RECQ4 disruption impairs restart of replication forks stalled by G4s. Overall, our findings highlight the essential roles of CST and RECQ4 in resolving G-rich regions, where they collaborate to resolve G4-induced replication deficiencies and maintain genomic homeostasis.
Assuntos
Replicação do DNA , Quadruplex G , Humanos , Proteínas de Ligação a Telômeros/genética , Homeostase do Telômero , Telômero/metabolismoRESUMO
CST (CTC1-STN1-TEN1) is a telomere associated complex that binds ssDNA and is required for multiple steps in telomere replication, including termination of G-strand extension by telomerase and synthesis of the complementary C-strand. CST contains seven OB-folds which appear to mediate CST function by modulating CST binding to ssDNA and the ability of CST to recruit or engage partner proteins. However, the mechanism whereby CST achieves its various functions remains unclear. To address the mechanism, we generated a series of CTC1 mutants and studied their effect on CST binding to ssDNA and their ability to rescue CST function in CTC1-/- cells. We identified the OB-B domain as a key determinant of telomerase termination but not C-strand synthesis. CTC1-ΔB expression rescued C-strand fill-in, prevented telomeric DNA damage signaling and growth arrest. However, it caused progressive telomere elongation and the accumulation of telomerase at telomeres, indicating an inability to limit telomerase action. The CTC1-ΔB mutation greatly reduced CST-TPP1 interaction but only modestly affected ssDNA binding. OB-B point mutations also weakened TPP1 association, with the deficiency in TPP1 interaction tracking with an inability to limit telomerase action. Overall, our results indicate that CTC1-TPP1 interaction plays a key role in telomerase termination.
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Telomerase , Humanos , Linhagem Celular , DNA de Cadeia Simples/genética , Mutação , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Sister chromatid cohesion (SCC), the pairing of sister chromatids after DNA replication until mitosis, is established by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. However, how SCC is established and maintained until mitosis remains incompletely understood, and emerging evidence suggests that replication stress may lead to premature SCC loss. Here, we report that the ssDNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. After depletion of CST subunits, we observed an increase in the complete loss of SCC. In addition, we determined that CST associates with the cohesin complex. Unexpectedly, we did not find evidence of altered cohesin loading or mitotic progression in the absence of CST; however, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Because replication stress was recently shown to induce SCC loss, we hypothesized that CST may be required to maintain or remodel SCC after DNA replication fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells after exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Acetilação , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Mitose , Ligação Proteica , Troca de Cromátide Irmã , CoesinasRESUMO
BACKGROUND: The comparative efficacy of various anticoagulation strategies has not been clearly established in patients with acute myocardial infarction who are undergoing percutaneous coronary intervention (PCI) according to current practice, which includes the use of radial-artery access for PCI and administration of potent P2Y12 inhibitors without the planned use of glycoprotein IIb/IIIa inhibitors. METHODS: In this multicenter, randomized, registry-based, open-label clinical trial, we enrolled patients with either ST-segment elevation myocardial infarction (STEMI) or non-STEMI (NSTEMI) who were undergoing PCI and receiving treatment with a potent P2Y12 inhibitor (ticagrelor, prasugrel, or cangrelor) without the planned use of glycoprotein IIb/IIIa inhibitors. The patients were randomly assigned to receive bivalirudin or heparin during PCI, which was performed predominantly with the use of radial-artery access. The primary end point was a composite of death from any cause, myocardial infarction, or major bleeding during 180 days of follow-up. RESULTS: A total of 6006 patients (3005 with STEMI and 3001 with NSTEMI) were enrolled in the trial. At 180 days, a primary end-point event had occurred in 12.3% of the patients (369 of 3004) in the bivalirudin group and in 12.8% (383 of 3002) in the heparin group (hazard ratio, 0.96; 95% confidence interval [CI], 0.83 to 1.10; P=0.54). The results were consistent between patients with STEMI and those with NSTEMI and across other major subgroups. Myocardial infarction occurred in 2.0% of the patients in the bivalirudin group and in 2.4% in the heparin group (hazard ratio, 0.84; 95% CI, 0.60 to 1.19; P=0.33), major bleeding in 8.6% and 8.6%, respectively (hazard ratio, 1.00; 95% CI, 0.84 to 1.19; P=0.98), definite stent thrombosis in 0.4% and 0.7%, respectively (hazard ratio, 0.54; 95% CI, 0.27 to 1.10; P=0.09), and death in 2.9% and 2.8%, respectively (hazard ratio, 1.05; 95% CI, 0.78 to 1.41; P=0.76). CONCLUSIONS: Among patients undergoing PCI for myocardial infarction, the rate of the composite of death from any cause, myocardial infarction, or major bleeding was not lower among those who received bivalirudin than among those who received heparin monotherapy. (Funded by the Swedish Heart-Lung Foundation and others; VALIDATE-SWEDEHEART ClinicalTrialsRegister.eu number, 2012-005260-10 ; ClinicalTrials.gov number, NCT02311231 .).
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Anticoagulantes/uso terapêutico , Heparina/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , Intervenção Coronária Percutânea , Idoso , Anticoagulantes/efeitos adversos , Terapia Combinada , Feminino , Hemorragia/induzido quimicamente , Heparina/administração & dosagem , Hirudinas/efeitos adversos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/terapia , Fragmentos de Peptídeos/efeitos adversos , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêuticoRESUMO
Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress.
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BACKGROUND: Timely percutaneous coronary intervention (PCI) improves prognosis in ST-elevation myocardial infarction (STEMI). However, recent reports indicate that patients with STEMI who present during non-regular working hours (off-hours) have a worse prognosis. The aim of this study was to compare outcome between patients with STEMI who underwent primary PCI during off-hours and regular hours. METHODS: We retrieved data from the Swedish Coronary Angiography and Angioplasty Registry (SCAAR) for all patients who underwent primary PCI in Region Västra Götaland due to STEMI between January 2004 and May 2013. We modeled unadjusted and adjusted Cox proportional-hazards regression and logistic regression models for the outcomes death, cardiogenic shock, stent thrombosis and in-stent restenosis. A propensity score-adjusted Cox proportional-hazards model, which adjusted for traditional cardiovascular risk factors was predefined as the primary statistical model. Death at any time during the study period was pre-specified as primary end-point. RESULTS: During the study period 4.611 (65%) patients underwent primary PCI due to STEMI during off-hours and 2,525 (35%) during regular hours. The risk of dying was similar among the groups for the primary endpoint death at any time during the study period (HR 1.00, 95% CI 0.89-1.12, P = 0.991) and for secondary end-point death within 30 days (HR 1.03; 95% CI 0.85-1.25, P = 0.735). The risks of developing cardiogenic shock, stent thrombosis, or in-stent restenosis were similar between the groups. CONCLUSIONS: In our region, short- and long-term prognosis for patients with STEMI who undergo primary PCI is similar for patients presenting during off-hours and regular hours.
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Plantão Médico , Acessibilidade aos Serviços de Saúde , Avaliação de Processos e Resultados em Cuidados de Saúde , Intervenção Coronária Percutânea , Infarto do Miocárdio com Supradesnível do Segmento ST/cirurgia , Idoso , Idoso de 80 Anos ou mais , Angiografia Coronária , Reestenose Coronária/etiologia , Trombose Coronária/etiologia , Bases de Dados Factuais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Intervenção Coronária Percutânea/efeitos adversos , Intervenção Coronária Percutânea/mortalidade , Sistema de Registros , Medição de Risco , Fatores de Risco , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico por imagem , Infarto do Miocárdio com Supradesnível do Segmento ST/mortalidade , Choque Cardiogênico/etiologia , Suécia , Fatores de Tempo , Resultado do TratamentoRESUMO
Mammalian CST (CTC1-STN1-TEN1) associates with telomeres and depletion of CTC1 or STN1 causes telomere defects. However, the function of mammalian CST remains poorly understood. We show here that depletion of CST subunits leads to both telomeric and non-telomeric phenotypes associated with DNA replication defects. Stable knockdown of CTC1 or STN1 increases the incidence of anaphase bridges and multi-telomeric signals, indicating genomic and telomeric instability. STN1 knockdown also delays replication through the telomere indicating a role in replication fork passage through this natural barrier. Furthermore, we find that STN1 plays a novel role in genome-wide replication restart after hydroxyurea (HU)-induced replication fork stalling. STN1 depletion leads to reduced EdU incorporation after HU release. However, most forks rapidly resume replication, indicating replisome integrity is largely intact and STN1 depletion has little effect on fork restart. Instead, STN1 depletion leads to a decrease in new origin firing. Our findings suggest that CST rescues stalled replication forks during conditions of replication stress, such as those found at natural replication barriers, likely by facilitating dormant origin firing.
Assuntos
Replicação do DNA , Proteínas de Ligação a Telômeros/genética , Telômero/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Humanos , Proteína 1 de Ligação a Repetições Teloméricas/genéticaRESUMO
BACKGROUND: Routine thrombus aspiration during primary percutaneous coronary intervention (PCI) in ST-elevation myocardial infarction (STEMI) did not reduce the primary composite endpoint in the "A Randomised Trial of Routine Aspiration ThrOmbecTomy With PCI Versus PCI ALone in Patients With STEMI Undergoing Primary PCI" (TOTAL) trial. We aimed to analyse a similar endpoint in "The Thrombus Aspiration in ST-Elevation myocardial infarction in Scandinavia" (TASTE) trial up to 180 days. METHODS: In TASTE, 7244 patients with STEMI were randomised to thrombus aspiration followed by PCI or to PCI alone. We analysed the quadruple composite endpoint of cardiovascular death, cardiogenic shock, rehospitalisation for myocardial infarction, or new hospitalisation for heart failure. Furthermore, an extended net-benefit composite endpoint including stent thrombosis, target vessel revascularization or stroke within 180 days was analysed. RESULTS: The primary quadruple composite endpoint occurred in 8.7 % (316 of 3621) in the thrombus aspiration group compared to 9.3 % (338 of 3623) in the PCI alone group (hazard ratio (HR), 0.93; 95 % confidence interval (CI); 0.80 - 1.09, P = 0.36) and the extended net-benefit composite endpoint in 12.0 % (436) vs. 13.2 % (479) (HR, 0.90; 95 % CI; 0.79 - 1.03, P = 0.12). Stroke within 30 days occurred in 0.7 % (27) vs. 0.7 % (24) (HR, 0.89; 95 % CI; 0.51-1.54, P = 0.68). CONCLUSIONS: A large and an extended composite endpoint analysis from the TASTE trial did not demonstrate any clinical benefit of routine thrombus aspiration during PCI in patients with STEMI. There was no evidence of an increased risk of stroke with thrombus aspiration.
Assuntos
Trombose Coronária/terapia , Infarto do Miocárdio/terapia , Intervenção Coronária Percutânea , Acidente Vascular Cerebral/etiologia , Trombectomia , Trombose Coronária/complicações , Trombose Coronária/diagnóstico , Trombose Coronária/mortalidade , Intervalo Livre de Doença , Insuficiência Cardíaca/etiologia , Humanos , Estimativa de Kaplan-Meier , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/mortalidade , Readmissão do Paciente , Intervenção Coronária Percutânea/efeitos adversos , Intervenção Coronária Percutânea/instrumentação , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Países Escandinavos e Nórdicos , Choque Cardiogênico/etiologia , Stents , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/mortalidade , Trombectomia/efeitos adversos , Fatores de Tempo , Resultado do TratamentoRESUMO
CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol ß, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a Telômeros , Humanos , Proteínas de Ligação a Telômeros/metabolismo , Proteínas de Ligação a Telômeros/genética , Telômero/metabolismo , Telômero/genética , DNA Glicosilases/metabolismo , DNA Glicosilases/genética , Estresse Oxidativo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Guanina/análogos & derivados , Guanina/metabolismo , DNA Polimerase beta/metabolismo , DNA Polimerase beta/genética , Reparo por ExcisãoRESUMO
Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, and individuals with HGPS seldom live beyond their mid-teens. The syndrome is commonly caused by a point mutation in the LMNA gene which codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The mutation causing HGPS leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs) and alterations in the nature of DSB repair. The source of DSBs in HGPS is often attributed to stalling and subsequent collapse of replication forks in conjunction with faulty recruitment of repair factors to damage sites. In this work, we used a model system involving immortalized human cell lines to investigate progerin-induced genomic damage. Using an immunofluorescence approach to visualize phosphorylated histone H2AX foci which mark sites of genomic damage, we report that cells engineered to express progerin displayed a significant elevation of endogenous damage in the absence of any change in the cell cycle profile or doubling time of cells. Genomic damage was enhanced and persistent in progerin-expressing cells treated with hydroxyurea. Overexpression of wild-type lamin A did not elicit the outcomes associated with progerin expression. Our results show that DNA damage caused by progerin can occur independently from global changes in replication or cell proliferation.
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BACKGROUND: The registry-based randomized VALIDATE-SWEDEHEART trial (NCT02311231) compared bivalirudin vs. heparin in patients undergoing percutaneous coronary intervention (PCI) for myocardial infarction (MI). It showed no difference in the composite primary endpoint of death, MI, or major bleeding at 180 days. Here, we report outcomes at two years. METHODS: Analysis of primary and secondary endpoints at two years of follow-up was prespecified in the study protocol. We report the study results for the extended follow-up time here. RESULTS: In total, 6006 patients were enrolled, 3005 with ST-segment elevation MI (STEMI) and 3001 with Non-STEMI (NSTEMI), representing 70 % of all eligible patients with these diagnoses during the study. The primary endpoint occurred in 14.0 % (421 of 3004) in the bivalirudin group compared with 14.3 % (429 of 3002) in the heparin group (hazard ratio [HR] 0.97; 95 % confidence interval [CI], 0.85-1.11; P = 0.70) at one year and in 16.7 % (503 of 3004) compared with 17.1 % (514 of 3002), (HR 0.97; 95 % CI, 0.96-1.10; P = 0.66) at two years. The results were consistent in patients with STEMI and NSTEMI and across major subgroups. CONCLUSIONS: Until the two-year follow-up, there were no differences in endpoints between patients with MI undergoing PCI and allocated to bivalirudin compared with those allocated to heparin. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02311231.
Assuntos
Anticoagulantes , Antitrombinas , Hemorragia , Heparina , Hirudinas , Infarto do Miocárdio sem Supradesnível do Segmento ST , Fragmentos de Peptídeos , Intervenção Coronária Percutânea , Proteínas Recombinantes , Sistema de Registros , Infarto do Miocárdio com Supradesnível do Segmento ST , Humanos , Hirudinas/efeitos adversos , Hirudinas/administração & dosagem , Heparina/efeitos adversos , Heparina/uso terapêutico , Heparina/administração & dosagem , Fragmentos de Peptídeos/uso terapêutico , Fragmentos de Peptídeos/efeitos adversos , Infarto do Miocárdio com Supradesnível do Segmento ST/terapia , Infarto do Miocárdio com Supradesnível do Segmento ST/mortalidade , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico , Intervenção Coronária Percutânea/efeitos adversos , Intervenção Coronária Percutânea/mortalidade , Proteínas Recombinantes/uso terapêutico , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/administração & dosagem , Masculino , Resultado do Tratamento , Feminino , Fatores de Tempo , Idoso , Pessoa de Meia-Idade , Antitrombinas/efeitos adversos , Antitrombinas/uso terapêutico , Antitrombinas/administração & dosagem , Anticoagulantes/efeitos adversos , Anticoagulantes/uso terapêutico , Hemorragia/induzido quimicamente , Infarto do Miocárdio sem Supradesnível do Segmento ST/terapia , Infarto do Miocárdio sem Supradesnível do Segmento ST/mortalidade , Infarto do Miocárdio sem Supradesnível do Segmento ST/diagnóstico , Infarto do Miocárdio sem Supradesnível do Segmento ST/diagnóstico por imagem , Fatores de Risco , SuéciaRESUMO
Background: Prognostic assessment of ventricular tachycardia (VT) or ventricular fibrillation (VF) in ST-segment elevation myocardial infarction (STEMI) is based mainly on distinguishing between early (<48 hours) and late arrhythmias, and does not take into account its time distribution with regard to reperfusion, or type of arrhythmia. Objective: We analyzed the prognostic value of early ventricular arrhythmias (VAs) in STEMI with regard to their type and timing. Methods: The prespecified analysis of the multicenter prospective Bivalirudin versus Heparin in ST-Segment and Non-ST-Segment Elevation Myocardial Infarctionin Patients on Modern Antiplatelet Therapy in the Swedish Web System for Enhancement and Development of Evidence-based Care in Heart Disease evaluated according to Recommended Therapies Registry Trial included 2886 STEMI patients undergoing primary percutaneous coronary intervention (PCI). VA episodes were characterized regarding their type and timing. Survival status at 180 days was assessed through the population registry. Results: Nonmonomorphic VT or VF was observed in 97 (3.4%) and monomorphic VT in 16 (0.5%) patients. Only 3 (2.7%) early VA episodes occurred after 24 hours from symptom onset. VA was associated with higher risk of death (hazard ratio 3.59; 95% confidence interval [CI] 2.01-6.42) after adjustment for age, sex, and STEMI localization. VA after PCI was associated with an increased mortality compared with VA before PCI (hazard ratio 6.68; 95% CI 2.90-15.41). Early VA was associated with in-hospital mortality (odds ratio 7.39; 95% CI 3.68-14.83) but not with long-term prognosis in patients discharged alive. The type of VA was not associated with mortality. Conclusion: VA after PCI was associated with an increased mortality compared with VA before PCI. Long-term prognosis did not differ between patients with monomorphic VT and nonmonomorphic VT or VF, but events were few. VA incidence during 24 to 48 hours of STEMI is negligibly low, thus precluding assessment of its prognostic importance.
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The mechanisms by which Streptococcus pneumoniae obtains carbohydrates for growth during airway colonization remain to be elucidated. The low concentration of free carbohydrates in the normal human airway suggests that pneumococci must utilize complex glycan structures for growth. The glycosaminoglycan hyaluronic acid is present on the apical surface of airway epithelial cells. As pneumococci express a hyaluronate lyase (Hyl) that cleaves hyaluronic acid into disaccharides, we hypothesized that during colonization pneumococci utilize the released carbohydrates for growth. Hyaluronic acid supported significant pneumococcal growth in an hyl-dependent manner. A phosphoenolpyruvate-dependent phosphotransferase system (PTS) and an unsaturated glucuronyl hydrolase (Ugl) encoded downstream of hyl are also essential for growth on hyaluronic acid. This genomic arrangement is present in several other organisms, suggesting conservation of the utilization mechanism between species. In vivo experiments support the hypothesis that S. pneumoniae utilizes hyaluronic acid as a carbon source during colonization. We also demonstrate that pneumococci can utilize the hyaluronic acid capsule of other bacterial species for growth, suggesting an alternative carbohydrate source for pneumococcal growth. Together, these data support a novel function for pneumococcal degradation of hyaluronic acid in vivo and provide mechanistic details of growth on this glycosaminoglycan.
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Ácido Hialurônico/metabolismo , Streptococcus pneumoniae/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , Camundongos , Fosfotransferases/metabolismo , Polissacarídeo-Liases/metabolismo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/crescimento & desenvolvimentoRESUMO
Chromosome end protection is essential to protect genome integrity. Telomeres, tracts of repetitive DNA sequence and associated proteins located at the chromosomal terminus, serve to safeguard the ends from degradation and unwanted double strand break repair. Due to the essential nature of telomeres in protecting the genome, a number of unique proteins have evolved to ensure that telomere length and structure are preserved. The inability to properly maintain telomeres can lead to diseases such as dyskeratosis congenita, pulmonary fibrosis and cancer. In this review, we will discuss the known functions of mammalian telomere-associated proteins, their role in telomere replication and length regulation and how these processes relate to genome instability and human disease.
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Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo , Telômero/fisiologia , Replicação do DNA , Instabilidade Genômica , Modelos Moleculares , Complexo Shelterina , Telomerase/metabolismo , Encurtamento do TelômeroRESUMO
Dna2 is a nuclease/helicase with proposed roles in DNA replication, double-strand break repair and telomere maintenance. For each role Dna2 is proposed to process DNA substrates with a 5'-flap. To date, however, Dna2 has not revealed a preference for binding or cleavage of flaps over single-stranded DNA. Using DNA binding competition assays we found that Dna2 has substrate structure specificity. The nuclease displayed a strong preference for binding substrates with a 5'-flap or some variations of flap structure. Further analysis revealed that Dna2 recognized and bound both the single-stranded flap and portions of the duplex region immediately downstream of the flap. A model is proposed in which Dna2 first binds to a flap base, and then the flap threads through the protein with periodic cleavage, to a terminal flap length of approximately 5 nt. This resembles the mechanism of flap endonuclease 1, consistent with cooperation of these two proteins in flap processing.
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DNA Helicases/metabolismo , DNA/química , Endodesoxirribonucleases/metabolismo , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Especificidade por SubstratoRESUMO
Generating long-lived mucosal and systemic antibodies through respiratory immunization with protective antigens encapsulated in nanoscale biodegradable particles could potentially decrease or eliminate the incidence of many infectious diseases, but requires the incorporation of a suitable mucosal immunostimulant. We previously found that respiratory immunization with a model protein antigen (LPS-free OVA) encapsulated in PLGA 50:50 nanoparticles (~380 nm diameter) surface-modified with complement peptide-derived immunostimulant 02 (CPDI-02; formerly EP67) through 2 kDa PEG linkers increases mucosal and systemic OVA-specific memory T-cells with long-lived surface phenotypes in young, naïve female C57BL/6 mice. Here, we determined if respiratory immunization with LPS-free OVA encapsulated in similar PLGA 50:50 microparticles (~1 µm diameter) surface-modified with CPDI-02 (CPDI-02-MP) increases long-term OVA-specific mucosal and systemic antibodies. We found that, compared to MP surface-modified with inactive, scrambled scCPDI-02 (scCPDI-02-MP), intranasal administration of CPDI-02-MP in 50 µL sterile PBS greatly increased titers of short-term (14 days post-immunization) and long-term (90 days post-immunization) antibodies against encapsulated LPS-free OVA in nasal lavage fluids, bronchoalveolar lavage fluids, and sera of young, naïve female C57BL/6 mice with minimal lung inflammation. Thus, surface modification of ~1 µm biodegradable microparticles with CPDI-02 is likely to increase long-term mucosal and systemic antibodies against encapsulated protein antigen after respiratory and possibly other routes of mucosal immunization.
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Serum tyrosine levels increase during aging, neurocognitive, metabolic, and cardiovascular disorders. However, calorie restriction (CR) and sleep lower serum tyrosine levels. We previously showed that tyrosine inhibits tyrosyl-tRNA synthetase (TyrRS)-mediated activation of poly-ADP-ribose polymerase 1 (PARP1). Here, we show that histone serine-ADP-ribosylation is decreased in Alzheimer's Disease (AD) brains, and increased tyrosine levels deplete TyrRS and cause neuronal DNA damage. However, dopamine and brain-derived neurotrophic factor (BDNF) increase TyrRS and histone serine-ADP-ribosylation. Furthermore, cis-resveratrol (cis-RSV) that binds to TyrRS mimicking a 'tyrosine-free' conformation increases TyrRS, facilitates histone serine-ADP-ribosylation-dependent DNA repair, and provides neuroprotection in a TyrRS-dependent manner. Conversely, trans-RSV that binds to TyrRS mimicking a 'tyrosine-like' conformation decreases TyrRS, inhibits serine-ADP-ribosylation-dependent DNA repair, and induces neurodegeneration in rat cortical neurons. Our findings suggest that age-associated increase in serum tyrosine levels may effect neurocognitive and metabolic disorders and offer a plausible explanation for divergent results obtained in clinical trials using resveratrol.
Assuntos
Histonas , Tirosina-tRNA Ligase , ADP-Ribosilação , Animais , Histonas/metabolismo , Ratos , Resveratrol/farmacologia , Serina/metabolismo , Tirosina/metabolismo , Tirosina-tRNA Ligase/metabolismoRESUMO
Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5'-3' endonuclease, the 5'-3' helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase alpha.
Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , Endonucleases Flap/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Humanos , Imunoprecipitação , Ligação ProteicaRESUMO
The regulation of DNA double-strand breaks (DSBs) and telomeres are diametrically opposed in the cell. DSBs are considered one of the most deleterious forms of DNA damage and must be quickly recognized and repaired. Telomeres, on the other hand, are specialized, stable DNA ends that must be protected from recognition as DSBs to inhibit unwanted chromosome fusions. Decisions to join DNA ends, or not, are therefore critical to genome stability. Yet, the processing of telomeres and DSBs share many commonalities. Accordingly, key decision points are used to shift DNA ends toward DSB repair vs. end protection. Additionally, DSBs can be repaired by two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of which repair pathway is employed is also dictated by a series of decision points that shift the break toward HR or NHEJ. In this review, we will focus on these decision points and the mechanisms that dictate end protection vs. DSB repair and DSB repair choice.
RESUMO
Few studies have examined the biosocial pathways linking socioeconomic status (SES) to accelerated aging in a population-based sample of southern US women. Even fewer have examined the importance of chronic compared to perceived stress in linking SES to women's salivary telomere length (STL). Using data from a probability-based sample of 156 US women and structural equation modeling, we examined three pathways - chronic stress exposure, stress appraisal, and coping behavior - linking SES to STL. SES was positively associated with STL (ßTE = 0.16, p < .05). Everyday discrimination was negatively associated with STL (ßDE = -0.21, p < .05), but perceived stress was positively associated with STL (ßDE = 0.20, p < .05). Current smoking decreased STL (ßDE = -0.19, p < .01). Perceived stress acted to suppress the negative relationship of chronic stress exposure on STL. Given the dearth of STL studies that include measures of both perceived and chronic stress, our study supports the importance of disentangling stress measures and a biosocial approach to the study of accelerated aging.