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1.
Nucleic Acids Res ; 49(20): 11653-11665, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34718732

RESUMO

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

3.
Nat Rev Mol Cell Biol ; 22(4): 283-298, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33564154

RESUMO

The regulation of telomere length in mammals is crucial for chromosome end-capping and thus for maintaining genome stability and cellular lifespan. This process requires coordination between telomeric protein complexes and the ribonucleoprotein telomerase, which extends the telomeric DNA. Telomeric proteins modulate telomere architecture, recruit telomerase to accessible telomeres and orchestrate the conversion of the newly synthesized telomeric single-stranded DNA tail into double-stranded DNA. Dysfunctional telomere maintenance leads to telomere shortening, which causes human diseases including bone marrow failure, premature ageing and cancer. Recent studies provide new insights into telomerase-related interactions (the 'telomere replisome') and reveal new challenges for future telomere structural biology endeavours owing to the dynamic nature of telomere architecture and the great number of structures that telomeres form. In this Review, we discuss recently determined structures of the shelterin and CTC1-STN1-TEN1 (CST) complexes, how they may participate in the regulation of telomere replication and chromosome end-capping, and how disease-causing mutations in their encoding genes may affect specific functions. Major outstanding questions in the field include how all of the telomere components assemble relative to each other and how the switching between different telomere structures is achieved.


Assuntos
Cromatina/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Cromossomos/metabolismo , DNA/metabolismo , Humanos , Telomerase/metabolismo
4.
Science ; 368(6495): 1081-1085, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32499435

RESUMO

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome's organization of double-stranded DNA.


Assuntos
Complexos Multiproteicos/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Telômero/química , Microscopia Crioeletrônica , DNA de Cadeia Simples/química , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteína de Replicação A/química
5.
Nat Commun ; 8(1): 1075, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-29057866

RESUMO

The human shelterin proteins associate with telomeric DNA to confer telomere protection and length regulation. They are thought to form higher-order protein complexes for their functions, but studies of shelterin proteins have been mostly limited to pairs of proteins. Here we co-express various human shelterin proteins and find that they form defined multi-subunit complexes. A complex harboring both TRF2 and POT1 has the strongest binding affinity to telomeric DNA substrates comprised of double-stranded DNA with a 3' single-stranded extension. TRF2 interacts with TIN2 with an unexpected 2:1 stoichiometry in the context of shelterin (RAP12:TRF22:TIN21:TPP11:POT11). Tethering of TPP1 to the telomere either via TRF2-TIN2 or via POT1 gives equivalent enhancement of telomerase processivity. We also identify a peptide region from TPP1 that is both critical and sufficient for TIN2 interaction. Our findings reveal new information about the architecture of human shelterin and how it performs its functions at telomeres.


Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Ligação Proteica , Telomerase/genética , Proteínas de Ligação a Telômeros/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
6.
Sci Rep ; 5: 18146, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-26657062

RESUMO

The bacterial nucleoid, a bacterial genome packed by nucleoid binding proteins, forms the physical basis for cellular processes such as gene transcription and DNA replication. Bacteria need to dynamically modulate their nucleoid structures at different growth phases and in response to environmental changes. At the nutrients deficient stationary phase, DNA-binding proteins from starved cells (Dps) and Integration host factors (IHF) are the two most abundant nucleoid associated proteins in E. coli. Yet, it remains unclear how the nucleoid architecture is controlled by the interplay between these two proteins, as well as the nucleoid's response to environmental changes. This question is addressed here using single DNA manipulation approach. Our results reveal that the two proteins are differentially selected for DNA binding, which can be tuned by changing environmental factors over physiological ranges including KCl (50-300 mM), MgCl2 (0-10 mM), pH (6.5-8.5) and temperature (23-37 °C). Increasing pH and MgCl2 concentrations switch from Dps-binding to IHF-binding. Stable Dps-DNA and IHF-DNA complexes are insensitive to temperature changes for the range tested. The environment dependent selection between IHF and Dps results in different physical organizations of DNA. Overall, our findings provide important insights into E. coli nucleoid architecture.


Assuntos
Empacotamento do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fatores Hospedeiros de Integração/genética , Ligação Competitiva/efeitos dos fármacos , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Concentração de Íons de Hidrogênio , Fatores Hospedeiros de Integração/metabolismo , Cloreto de Magnésio/farmacologia , Magnetismo , Pinças Ópticas , Cloreto de Potássio/farmacologia , Ligação Proteica/efeitos dos fármacos
7.
Artigo em Inglês | MEDLINE | ID: mdl-26382432

RESUMO

Toroid formation is an important mechanism for DNA condensation in cells. The length change during DNA condensation was investigated in previous single-molecule experiments. However, DNA twist is key to understanding the topological kinetics of DNA condensation. In this study, DNA twist as well as DNA length was traced during the DNA condensation by the freely orbiting magnetic tweezers and the tilted magnetic tweezers combined with Brownian dynamics simulations. The experimental results disclose the complex relationship between DNA extension and backbone rotation. Brownian dynamics simulations show that the toroid formation follows a wiggling pathway which leads to the complex DNA backbone rotation as revealed in our experiments. These findings provide the complete description of multivalent cation-dependent DNA toroid formation under tension.


Assuntos
DNA/química , Conformação de Ácido Nucleico , Simulação por Computador , Digoxigenina/química , Cinética , Modelos Lineares , Modelos Químicos , Modelos Genéticos , Rotação , Eletricidade Estática , Estreptavidina/química
8.
Nucleic Acids Res ; 42(13): 8789-95, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25013179

RESUMO

As critical DNA structures capping the human chromosome ends, the stability and structural polymorphism of human telomeric G-quadruplex (G4) have drawn increasing attention in recent years. This work characterizes the equilibrium transitions of single-molecule telomeric G4 at physiological K(+) concentration. We report three folded states of telomeric G4 with markedly different lifetime and mechanical stability. Our results show that the kinetically favored folding pathway is through a short-lived intermediate state to a longer-lived state. By examining the force dependence of transition rates, the force-dependent transition free energy landscape for this pathway is determined. In addition, an ultra-long-lived form of telomeric G4 structure with a much stronger mechanical stability is identified.


Assuntos
Quadruplex G , Telômero/química , Fenômenos Biomecânicos , Humanos , Cinética
9.
Nucleic Acids Res ; 42(13): 8369-78, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24990375

RESUMO

The Escherichia coli H-NS protein is a major nucleoid-associated protein that is involved in chromosomal DNA packaging and gene regulatory functions. These biological processes are intimately related to the DNA supercoiling state and thus suggest a direct relationship between H-NS binding and DNA supercoiling. Here, we show that H-NS, which has two distinct DNA-binding modes, is able to differentially regulate DNA supercoiling. H-NS DNA-stiffening mode caused by nucleoprotein filament formation is able to suppress DNA plectoneme formation during DNA supercoiling. In contrast, when H-NS is in its DNA-bridging mode, it is able to promote DNA plectoneme formation during DNA supercoiling. In addition, the DNA-bridging mode is able to block twists diffusion thus trapping DNA in supercoiled domains. Overall, this work reveals the mechanical interplay between H-NS and DNA supercoiling which provides insights to H-NS organization of chromosomal DNA based on its two distinct DNA architectural properties.


Assuntos
DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Fenômenos Biomecânicos , Ligação Proteica
10.
Nucleic Acids Res ; 41(10): 5263-72, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23580555

RESUMO

Bacterial nucleoid-associated proteins, such as H-NS-like proteins in Enterobacteriaceae, are abundant DNA-binding proteins that function in chromosomal DNA organization and gene transcription regulation. The Mycobacterium tuberculosis Lsr2 protein has been proposed to be the first identified H-NS analogue in Gram-positive bacteria based on its capability to complement numerous in vivo functions of H-NS. Here, we report that Lsr2 cooperatively binds to DNA forming a rigid Lsr2 nucleoprotein complex that restricts DNA accessibility, similar to H-NS. On large DNA, the rigid Lsr2 nucleoprotein complexes can mediate DNA condensation into highly compact DNA conformations. In addition, the responses of Lsr2 nucleoprotein complex to environmental factors (salt concentration, temperature and pH) were studied over physiological ranges. These results provide mechanistic insights into how Lsr2 may mediate its gene silencing, genomic DNA protection and organization functions in vivo. Finally, our results strongly support that Lsr2 is an H-NS-like protein in Gram-positive bacteria from a structural perspective.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/efeitos dos fármacos , DNA/metabolismo , Proteínas de Ligação a DNA/química , Concentração de Íons de Hidrogênio , Cloreto de Potássio/farmacologia , Temperatura
11.
Nucleic Acids Res ; 41(2): 746-53, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23180762

RESUMO

Dan is a transcription factor that regulates the ttd operon encoding tartrate dehydratase. During anaerobic conditions, its copy number increases by 100-fold, making Dan an abundant nucleoid-associated protein. However, little is known about the mode of Dan-DNA interaction. To understand its cellular functions, we used single-molecule manipulation and imaging techniques to show that Dan binds cooperatively along DNA, resulting in formation of a rigid periodic nucleoprotein filament that strongly restricts accessibility to DNA. Furthermore, in the presence of physiologic levels of magnesium, these filaments interact with each other to cause global DNA condensation. Overall, these results shed light on the architectural role of Dan in the compaction of Escherichia coli chromosomal DNA under anaerobic conditions. Formation of the nucleoprotein filament provides a basis in understanding how Dan may play roles in both chromosomal DNA protection and gene regulation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , DNA/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Nucleoproteínas/metabolismo , Nucleoproteínas/ultraestrutura , Anaerobiose , Cromossomos Bacterianos , DNA/metabolismo , DNA/ultraestrutura , Escherichia coli/genética , Cloreto de Magnésio/química
12.
Sci Rep ; 2: 509, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22798986

RESUMO

H-NS is an abundant nucleoid-associated protein in bacteria that globally silences genes, including horizontally-acquired genes related to pathogenesis. Although it has been shown that H-NS has multiple modes of DNA-binding, which mode is employed in gene silencing is still unclear. Here, we report that in H-NS mutants that are unable to silence genes, are unable to form a rigid H-NS nucleoprotein filament. These results indicate that the H-NS nucleoprotein filament is crucial for its gene silencing function, and serves as the fundamental structural basis for gene silencing by H-NS and likely other H-NS-like bacterial proteins.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Nucleoproteínas/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/ultraestrutura , Microscopia de Força Atômica , Modelos Biológicos , Mutação
13.
Nucleic Acids Res ; 40(8): 3316-28, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22187157

RESUMO

Nucleoid-associated proteins are bacterial proteins that are responsible for chromosomal DNA compaction and global gene regulation. One such protein is Escherichia coli Histone-like nucleoid structuring protein (H-NS) which functions as a global gene silencer. Whereas the DNA-binding mechanism of H-NS is well-characterized, its paralogue, StpA which is also able to silence genes is less understood. Here we show that StpA is similar to H-NS in that it is able to form a rigid filament along DNA. In contrast to H-NS, the StpA filament interacts with a naked DNA segment to cause DNA bridging which results in simultaneous stiffening and bridging of DNA. DNA accessibility is effectively blocked after the formation of StpA filament on DNA, suggesting rigid filament formation is the important step in promoting gene silencing. We also show that >1 mM magnesium promotes higher order DNA condensation, suggesting StpA may also play a role in chromosomal DNA packaging.


Assuntos
Proteínas de Ligação a DNA/ultraestrutura , DNA/química , Proteínas de Escherichia coli/ultraestrutura , Chaperonas Moleculares/ultraestrutura , DNA/metabolismo , DNA/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Inativação Gênica , Cinética , Magnésio/química , Microscopia de Força Atômica , Chaperonas Moleculares/metabolismo , Conformação de Ácido Nucleico , Cloreto de Potássio/química
14.
Appl Microbiol Biotechnol ; 71(5): 728-35, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16283294

RESUMO

This paper reports the successful isolation and characterization of a new phenol-degrading bacterium, strain EDP3, from activated sludge. Strain EDP3 is a nonmotile, strictly aerobic, Gram-negative, and short-rod or coccobacillary bacterium, which occurs singly, in pairs, or in clusters. 16S rRNA gene sequence analysis revealed that strain EDP3 belonged to the gamma group of Proteobacteria, with a 97.0% identity to 16S rRNA gene sequences of Acinetobacter calcoaceticus. Strain EDP3 could aerobically grow on a number of aromatic compounds, such as phenol, sodium benzoate, p-hydroxybenzoate, phenylacetate, benzene, ethylbenzene, benzylalcohol, and so on. In particular, it could mineralize up to 1,000 mg l(-1) phenol at room temperature (25 degrees C). The growth kinetics of strain EDP3 on phenol as a sole carbon and energy source at 25 degrees C can be described using the Haldane equation. It has a maximal specific growth rate (mu(max)) of 0.28 h(-1), a half-saturation constant (K(S)) of 1,167.1 mg l(-1), and a substrate inhibition constant (Ki) of 58.5 mg l(-1). Values of yield coefficient (Y(X/S)) are between 0.4 and 0.6 mg dry cell (mg phenol)(-1). Strain EDP3 has high tolerance to the toxicity of phenol (up to 1,000 mg l(-1)). It therefore could be an excellent candidate for the biotreatment of high-strength phenol-containing industrial wastewaters and for the in situ bioremediation of phenol-contaminated soils.


Assuntos
Acinetobacter calcoaceticus/classificação , Acinetobacter calcoaceticus/isolamento & purificação , Resíduos Industriais , Fenol/metabolismo , Esgotos/microbiologia , Acinetobacter calcoaceticus/genética , Acinetobacter calcoaceticus/metabolismo , Aerobiose , Biodegradação Ambiental , Meios de Cultura , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
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