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1.
Artigo em Inglês | MEDLINE | ID: mdl-35006640

RESUMO

We report a convenient method for benzylic H/D exchange of a wide variety of substrates bearing primary, secondary, or tertiary C-H bonds via a reversible η 6 -coordination strategy. A doubly cationic [Cp CF3 Rh(III)] 2+ catalyst that serves as an arenophile facilitates deprotonation of inert benzylic hydrogen atoms (p K a > 40 in DMSO) without affecting other hydrogen atoms, such as those on aromatic rings or in α -positions of carboxylate groups. Notably, the H/D exchange reactions feature high stereoretention. We demonstrated the potential utility of this method by using it for deuterium labeling of ten pharmaceuticals and their analogues.

2.
J Am Chem Soc ; 2022 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-35015956

RESUMO

Given the wide prevalence and ready availability of both phenols and amines, aniline synthesis through direct coupling between these starting materials would be extremely attractive. Herein, we describe a rhodium-catalyzed amination of phenols, which provides concise access to diverse anilines, with water as the sole byproduct. The arenophilic rhodium catalyst facilitates the inherently difficult keto-enol tautomerization of phenols by means of π-coordination, allowing for the subsequent dehydrative condensation with amines. We demonstrate the generality of this redox-neutral catalysis by carrying out reactions of a large array of phenols with various electronic properties and a wide variety of primary and secondary amines. Several examples of late-stage functionalization of structurally complex bioactive molecules, including pharmaceuticals, further illustrate the potential broad utility of the method.

3.
Signal Transduct Target Ther ; 6(1): 405, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34795208

RESUMO

Thalidomide induces γ-globin expression in erythroid progenitor cells, but its efficacy on patients with transfusion-dependent ß-thalassemia (TDT) remains unclear. In this phase 2, multi-center, randomized, double-blind clinical trial, we aimed to determine the safety and efficacy of thalidomide in TDT patients. A hundred patients of 14 years or older were randomly assigned to receive placebo or thalidomide for 12 weeks, followed by an extension phase of at least 36 weeks. The primary endpoint was the change of hemoglobin (Hb) level in the patients. The secondary endpoints included the red blood cell (RBC) units transfused and adverse effects. In the placebo-controlled period, Hb concentrations in patients treated with thalidomide achieved a median elevation of 14.0 (range, 2.5 to 37.5) g/L, whereas Hb in patients treated with placebo did not significantly change. Within the 12 weeks, the mean RBC transfusion volume for patients treated with thalidomide and placebo was 5.4 ± 5.0 U and 10.3 ± 6.4 U, respectively (P < 0.001). Adverse events of drowsiness, dizziness, fatigue, pyrexia, sore throat, and rash were more common with thalidomide than placebo. In the extension phase, treatment with thalidomide for 24 weeks resulted in a sustainable increase in Hb concentrations which reached 104.9 ± 19.0 g/L, without blood transfusion. Significant increase in Hb concentration and reduction in RBC transfusions were associated with non ß0/ß0 and HBS1L-MYB (rs9399137 C/T, C/C; rs4895441 A/G, G/G) genotypes. These results demonstrated that thalidomide is effective in patients with TDT.

4.
Nat Commun ; 12(1): 5881, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620857

RESUMO

Hydroalkylation, the direct addition of a C(sp3)-H bond across an olefin, is a desirable strategy to produce valuable, complex structural motifs in functional materials, pharmaceuticals, and natural products. Herein, we report a reliable method for accessing α-branched amines via nickel-catalyzed hydroalkylation reactions. Specifically, by using bis(cyclooctadiene)nickel (Ni(cod)2) together with a phosphine ligand, we achieved a formal C(sp3)-H bond insertion reaction between olefins and N-sulfonyl amines without the need for an external hydride source. The amine not only provides the alkyl motif but also delivers hydride to the olefin by means of a nickel-engaged ß-hydride elimination/reductive elimination process. This method provides a platform for constructing chiral α-branched amines by using a P-chiral ligand, demonstrating its potential utility in organic synthesis. Notably, a sulfonamidyl boronate complex formed in situ under basic conditions promotes ring-opening of the azanickellacycle reaction intermediate, leading to a significant improvement of the catalytic efficiency.

5.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064786

RESUMO

Single-molecule imaging is emerging as a revolutionary approach to studying fundamental questions in plants. However, compared with its use in animals, the application of single-molecule imaging in plants is still underexplored. Here, we review the applications, advantages, and challenges of single-molecule fluorescence imaging in plant systems from the perspective of methodology. Firstly, we provide a general overview of single-molecule imaging methods and their principles. Next, we summarize the unprecedented quantitative details that can be obtained using single-molecule techniques compared to bulk assays. Finally, we discuss the main problems encountered at this stage and provide possible solutions.


Assuntos
Fenômenos Fisiológicos Vegetais , Plantas/metabolismo , Imagem Individual de Molécula/métodos
6.
J Phys Chem Lett ; 12(13): 3361-3366, 2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33783224

RESUMO

Single-stranded guanine-rich RNA sequences have a propensity to fold into compact G-quadruplexes (RG4s). The conformational transitions of these molecules provide an important way to regulate their biological functions. Here, we examined the stability and conformation of an RG4-forming sequence identified near the end of human telomerase RNA. We found that a proximal single-stranded (ss) RNA significantly impairs RG4 stability at physiological K+ concentrations, resulting in a reduced RG4 rupture force of ∼ 24.4 pN and easier accessibility of the G-rich sequence. The destabilizing effect requires a minimum of six nucleotides of ssRNA and is effective at either end of RG4. Remarkably, this RG4-forming sequence, under the influence of such a proximal ssRNA, exhibits interconversions between at least three less stable RG4 conformers that might represent potential intermediates along its folding/unfolding pathway. This work provides insights into the stability and folding dynamics of RG4 that are essential for understanding its biological functions.


Assuntos
Quadruplex G , RNA/química , Telomerase/química , Corantes Fluorescentes/química , Humanos , Íons/química , Conformação de Ácido Nucleico , Potássio/química , RNA/genética , Estabilidade de RNA , Telomerase/genética
7.
Anal Bioanal Chem ; 412(27): 7395-7404, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32851458

RESUMO

G-Quadruplexes (G4s) are thermodynamically stable, compact, and poorly hydrated structures that pose a potent obstacle for chromosome replication and gene expression, and requiring resolution by helicases in a cell. Bulk stopped-flow fluorescence assays have provided many mechanistic insights into helicase-mediated duplex DNA unwinding. However, to date, detailed studies on intramolecular G-quadruplexes similar or comparable with those used for studying duplex DNA are still lacking. Here, we describe a method for the direct and quantitative measurement of helicase-mediated intramolecular G-quadruplex unfolding in real time. We designed a series of site-specific fluorescently double-labeled intramolecular G4s and screened appropriate substrates to characterize the helicase-mediated G4 unfolding. With the developed method, we determined, for the first time to our best knowledge, the unfolding and refolding constant of G4 (≈ 5 s-1), and other relative parameters under single-turnover experimental conditions in the presence of G4 traps. Our approach not only provides a new paradigm for characterizing helicase-mediated intramolecular G4 unfolding using stopped-flow assays but also offers a way to screen for inhibitors of G4 unfolding helicases as therapeutic drug targets. Graphical abstract.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/metabolismo , Ensaios Enzimáticos/métodos , Quadruplex G , RecQ Helicases/metabolismo , Animais , DNA/química , DNA/metabolismo , Drosophila/enzimologia , Humanos , Cinética , Espectrometria de Fluorescência/métodos , Especificidade por Substrato
8.
Int J Biol Macromol ; 164: 902-910, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32693146

RESUMO

RecD family helicases play an important role in prokaryotic genome stability and serve as the structural models for studying superfamily 1B (SF1B) helicases. However, RecD-catalyzed duplex DNA unwinding behavior and the underlying mechanism are still elusive. RecD family helicases share a common proto-helicase with eukaryotic Pif1 family helicases, which are well known for their outstanding G-quadruplex (G4) unwinding ability. However, there are still controversial points as to whether and how RecD helicases unfold G4 structures. Here, single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers (MT) were used to study Deinococcus radiodurans RecD2 (DrRecD2)-mediated duplex DNA unwinding and resolution of G4 structures. A symmetric, repetitive unwinding phenomenon was observed on duplex DNA, revealed from the strand switch and translocation of one monomer. Furthermore, we found that DrRecD2 was able to unwind both parallel and antiparallel G4 structures without obvious topological preferences. Surprisingly, the unwinding properties of RecD on duplex and G4 DNA are different from those of Pif1. The findings provide an example, in which the patterns of two molecules derived from a common ancestor deviate during evolution, and they are of significance for understanding the unwinding mechanism and function of SF1B helicases.


Assuntos
Proteínas de Bactérias/química , DNA Helicases/química , Deinococcus/enzimologia , Quadruplex G , Proteínas de Saccharomyces cerevisiae/química , Catálise , Dicroísmo Circular , DNA de Cadeia Simples/química , Transferência Ressonante de Energia de Fluorescência/métodos , Instabilidade Genômica , Magnetismo
9.
J Biol Chem ; 295(16): 5461-5469, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184352

RESUMO

Telomeres are specific nucleoprotein structures that are located at the ends of linear eukaryotic chromosomes and play crucial roles in genomic stability. Telomere DNA consists of simple repeats of a short G-rich sequence: TTAGGG in mammals and TTTAGGG in most plants. In recent years, the mammalian telomeric G-rich repeats have been shown to form G-quadruplex (G4) structures, which are crucial for modulating telomere functions. Surprisingly, even though plant telomeres are essential for plant growth, development, and environmental adaptions, only few reports exist on plant telomeric G4 DNA (pTG4). Here, using bulk and single-molecule assays, including CD spectroscopy, and single-molecule FRET approaches, we comprehensively characterized the structure and dynamics of a typical plant telomeric sequence, d[GGG(TTTAGGG)3]. We found that this sequence can fold into mixed G4s in potassium, including parallel and antiparallel structures. We also directly detected intermediate dynamic transitions, including G-hairpin, parallel G-triplex, and antiparallel G-triplex structures. Moreover, we observed that pTG4 is unfolded by the AtRecQ2 helicase but not by AtRecQ3. The results of our work shed light on our understanding about the existence, topological structures, stability, intermediates, unwinding, and functions of pTG4.


Assuntos
Cromossomos de Plantas/genética , Quadruplex G , Telômero/química , Proteínas de Arabidopsis/metabolismo , Instabilidade Genômica , RecQ Helicases/metabolismo , Telômero/genética
10.
Biochemistry ; 58(38): 3955-3959, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31468963

RESUMO

Guanine (G) quadruplexes (G4s) can be formed by G-rich sequences when stabilized by the binding of cations (typically K+ or Na+) and play an essential role in replication, recombination, transcription, and telomere maintenance. Understanding of the G4 folding process is crucial for determining their cellular functions. However, G4-K+ interactions and folding pathways are still not well understood. By using human telomeric G4 (hTG4) as an example, two binding states corresponding to two K+ cations binding to hTG4 were distinguished clearly and fitted precisely. The basic binding parameters during G4-K+ interactions were measured and calculated by taking advantage of microscale thermophoresis (MST), which monitors the changes in charge and size at the same time. The G-hairpin and G-triplex have been suggested as intermediates during G4 folding and unfolding. We further analyzed the equilibrium dissociation constants of 10 possible folding intermediates using MST; thus, the energetically favorable folding/unfolding pathways were proposed. The results might not only shed new light on G4-K+ interactions and G4 folding pathways but also provide an example for experimentally studying DNA-ion interactions.


Assuntos
DNA/química , Quadruplex G , Potássio/química , Telômero/química , Cátions Monovalentes/química , Dicroísmo Circular , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Cinética , Oligonucleotídeos/química , Coloração e Rotulagem , Termodinâmica
11.
ACS Synth Biol ; 8(7): 1663-1672, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31264849

RESUMO

Despite having a great variety of topologies, most DNA, RNA, and RNA-DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.


Assuntos
Bioensaio/métodos , DNA de Cadeia Simples/genética , DNA/genética , RNA/genética , Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Hibridização de Ácido Nucleico/métodos , Pinças Ópticas
13.
Biochemistry ; 58(15): 2009-2016, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30900876

RESUMO

S-DNA (stretched DNA) is an elongated base-paired DNA conformation under high tension. Because the RecA/Rad51 family DNA recombinases form helical filaments on DNA and mediate the formation of the DNA triplex (D-loop), in which the DNA is stretched, and because the extension of these nucleoprotein filaments is similar to the extension of S-DNA, S-DNA has long been hypothesized as a possible state of DNA that participants in RecA/Rad51-mediated DNA strand exchange in homologous recombination. Such a hypothesis, however, is still lacking direct experimental studies. In this work, we have studied the polymerization and strand exchange on S-DNA mediated by Escherichia coli RecA, human Rad51, and Saccharomyces cerevisiae Rad51 by single-molecule magnetic tweezers. We report that RecA/Rad51 polymerizes faster on S-DNA than on B-DNA with the same buffer conditions. Furthermore, the RecA/Rad51-mediated DNA triplex forms faster from S-DNA than from B-DNA together with the homologous single-stranded DNA. These results provide evidence that S-DNA can interact with RecA and Rad51 and shed light on the possible functions of S-DNA.


Assuntos
Pareamento de Bases , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Escherichia coli/química , Rad51 Recombinase/química , Recombinases Rec A/química , Proteínas de Saccharomyces cerevisiae/química , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Conformação de Ácido Nucleico , Polimerização , Ligação Proteica , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Mecânico
14.
Structure ; 26(3): 403-415.e4, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29429875

RESUMO

Helicase DHX36 plays essential roles in cell development and differentiation at least partially by resolving G-quadruplex (G4) structures. Here we report crystal structures of the Drosophila homolog of DHX36 (DmDHX36) in complex with RNA and a series of DNAs. By combining structural, small-angle X-ray scattering, molecular dynamics simulation, and single-molecule fluorescence studies, we revealed that positively charged amino acids in RecA2 and OB-like domains constitute an elaborate structural pocket at the nucleic acid entrance, in which negatively charged G4 DNA is tightly bound and partially destabilized. The G4 DNA is then completely unfolded through the 3'-5' translocation activity of the helicase. Furthermore, crystal structures and DNA binding assays show that G-rich DNA is preferentially recognized and in the presence of ATP, specifically bound by DmDHX36, which may cooperatively enhance the G-rich DNA translocation and G4 unfolding. On the basis of these results, a conceptual G4 DNA-resolving mechanism is proposed.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , DNA/química , Drosophila/metabolismo , RNA/química , Animais , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Drosophila/química , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Quadruplex G , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Desdobramento de Proteína , RNA/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
15.
Nucleic Acids Res ; 46(3): 1486-1500, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29202194

RESUMO

The Saccharomyces cerevisiae Pif1 protein (ScPif1p) is the prototypical member of the Pif1 family of DNA helicases. ScPif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and suppresses genome instability at G-quadruplex motifs. Here, we report the crystal structures of a truncated ScPif1p (ScPif1p237-780) in complex with different ssDNAs. Our results have revealed that a yeast-specific insertion domain protruding from the 2B domain folds as a bundle bearing an α-helix, α16. The α16 helix regulates the helicase activities of ScPif1p through interactions with the previously identified loop3. Furthermore, a biologically relevant dimeric structure has been identified, which can be further specifically stabilized by G-quadruplex DNA. Basing on structural analyses and mutational studies with DNA binding and unwinding assays, a potential G-quadruplex DNA binding site in ScPif1p monomers is suggested. Our results also show that ScPif1p uses the Q-motif to preferentially hydrolyze ATP, and a G-rich tract is preferentially recognized by more residues, consistent with previous biochemical observations. These findings provide a structural and mechanistic basis for understanding the multifunctional ScPif1p.


Assuntos
Trifosfato de Adenosina/química , DNA Helicases/química , DNA Fúngico/química , DNA de Cadeia Simples/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Quadruplex G , Expressão Gênica , Hidrólise , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica
16.
Nucleic Acids Res ; 45(19): 11401-11412, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-28977514

RESUMO

G-quadruplex (G4) can be formed by G-rich DNA sequences that are widely distributed throughout the human genome. Although G-triplex and G-hairpin have been proposed as G4 folding intermediates, their formation still requires further investigation by experiments. Here, we employed single-molecule FRET to characterize the folding dynamics of G4 from human telomeric sequence. First, we observed four states during G4 folding initially assigned to be anti-parallel G4, G-triplex, G-hairpin and unfolded ssDNA. Then we constructed putative intra-strand G-triplex, G-hairpin structures and confirmed their existences in both NaCl and KCl. Further studies revealed those structures are going through dynamic transitions between different states and show relatively weak dependence on cations, unlike G4. Based on those results and molecular dynamics simulations, we proposed a multi-pathway folding mechanism for human telomeric G4. The present work may shed new light on our current understanding about the existence and stability of G4 intermediate states.


Assuntos
DNA/química , Quadruplex G , Conformação de Ácido Nucleico , Telômero/genética , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Transdução de Sinais/genética
17.
Sci Rep ; 7: 43954, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28266653

RESUMO

Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase's DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning.


Assuntos
Galinhas , DNA Helicases/metabolismo , DNA/metabolismo , Helicase da Síndrome de Werner/metabolismo , Animais , Transferência Ressonante de Energia de Fluorescência , Imagem Individual de Molécula
18.
Nucleic Acids Res ; 44(17): 8385-94, 2016 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-27471032

RESUMO

Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a 'waiting time'. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells.


Assuntos
Biocatálise , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Quadruplex G , Desnaturação de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Replicação do DNA , Multimerização Proteica , Especificidade por Substrato , Fatores de Tempo
19.
Nucleic Acids Res ; 43(9): 4614-26, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25897130

RESUMO

Mutations in the RecQ DNA helicase gene BLM give rise to Bloom's syndrome, which is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition. BLM helicase is highly active in binding and unwinding G-quadruplexes (G4s), which are physiological targets for BLM, as revealed by genome-wide characterizations of gene expression of cells from BS patients. With smFRET assays, we studied the molecular mechanism of BLM-catalyzed G4 unfolding and showed that ATP is required for G4 unfolding. Surprisingly, depending on the molecular environments of G4, BLM unfolds G4 through different mechanisms: unfolding G4 harboring a 3'-ssDNA tail in three discrete steps with unidirectional translocation, and unfolding G4 connected to dsDNA by ssDNA in a repetitive manner in which BLM remains anchored at the ss/dsDNA junction, and G4 was unfolded by reeling in ssDNA. This indicates that one BLM molecule may unfold G4s in different molecular environments through different mechanisms.


Assuntos
DNA/química , Quadruplex G , RecQ Helicases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Transferência Ressonante de Energia de Fluorescência
20.
Biochem J ; 466(1): 189-99, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25471447

RESUMO

Recent advances in G-quadruplex (G4) studies have confirmed that G4 structures exist in living cells and may have detrimental effects on various DNA transactions. How helicases resolve G4, however, has just begun to be studied and remains largely unknown. In the present paper, we use single-molecule fluorescence assays to probe Pif1-catalysed unfolding of G4 in a DNA construct resembling an ongoing synthesis of lagging strand stalled by G4. Strikingly, Pif1 unfolds and then halts at the ss/dsDNA junction, followed by rapid reformation of G4 and 'acrobatic' re-initiation of unfolding by the same monomer. Thus, Pif1 unfolds single G4 structures repetitively. Furthermore, it is found that Pif1 unfolds G4 sequentially in two large steps. Our study has revealed that, as a stable intermediate, G-triplex (G3) plays an essential role in this process. The repetitive unfolding activity may facilitate Pif1 disrupting the continuously reforming obstructive G4 structures to rescue a stalled replication fork. The proposed mechanism for step-wise unfolding of G4 is probably applicable to other helicases that resolve G4 structures for maintaining genome stability.


Assuntos
DNA Helicases/química , DNA de Cadeia Simples/química , Quadruplex G , Genoma Fúngico , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Biocatálise , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Expressão Gênica , Instabilidade Genômica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Coloração e Rotulagem
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