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1.
J Biol Chem ; 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350334

RESUMO

Replication protein A (RPA) is the major eukaryotic ssDNA-binding protein and has essential roles in genome maintenance. RPA binds to ssDNA through multiple modes, and recent studies have suggested that the RPA-ssDNA interaction is dynamic. However, how RPA alternates between different binding modes and modifies ssDNA structures in this dynamic interaction remains unknown. Here, we used single-molecule FRET to systematically investigate the interaction between human RPA and ssDNA. We show that RPA can adopt different types of binding complexes with ssDNAs of different lengths, leading to the straightening or bending of the ssDNAs, depending on both the length and structure of the ssDNA substrate and the RPA concentration. Importantly, we noted that some of the complexes are highly dynamic, whereas others appear relatively static. On the basis of the above observations, we propose a model explaining how RPA dynamically engages with ssDNA. Of note, fluorescence anisotropy indicated that RPA can also associate with RNA, but with a lower binding affinity than with ssDNA. At the single-molecule level, we observed that RPA is undergoing rapid and repetitive associations with and dissociation from the RNA. This study may provide new insights into the rich dynamics of RPA binding to ssDNA and RNA.

2.
J Biol Chem ; 293(48): 18504-18513, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30305390

RESUMO

G-quadruplexes (G4s) are four-stranded DNA structures formed by Hoogsteen base pairing between stacked sets of four guanines. Pif1 helicase plays critical roles in suppressing genomic instability in the yeast Saccharomyces cerevisiae by resolving G4s. However, the structural properties of G4s in S. cerevisiae and the substrate preference of Pif1 for different G4s remain unknown. Here, using CD spectroscopy and 83 G4 motifs from S. cerevisiae ranging in length from 30 to 60 nucleotides, we first show that G4 structures can be formed with a broad range of loop sizes in vitro and that a parallel conformation is favored. Using single-molecule FRET analysis, we then systematically addressed Pif1-mediated unwinding of various G4s and found that Pif1 is sensitive to G4 stability. Moreover, Pif1 preferentially unfolded antiparallel G4s rather than parallel G4s having similar stability. Furthermore, our results indicate that most G4 structures in S. cerevisiae sequences have long loops and can be efficiently unfolded by Pif1 because of their low stability. However, we also found that G4 structures with short loops can be barely unfolded. This study highlights the formidable capability of Pif1 to resolve the majority of G4s in S. cerevisiae sequences, narrows the fractions of G4s that may be challenging for genomic stability, and provides a framework for understanding the influence of different G4s on genomic stability via their processing by Pif1.

3.
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.

4.
FEBS J ; 284(23): 4051-4065, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28986969

RESUMO

Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli.


Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/metabolismo , Quadruplex G , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Modelos Genéticos , Modelos Moleculares , Conformação de Ácido Nucleico
5.
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
6.
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
7.
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
8.
ACS Omega ; 1(2): 244-250, 2016 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-30023477

RESUMO

G-Quadruplex DNA structure has been proven to be a binding target for small molecular organic compounds and hence regarded as a promising pharmacological target. Cisplatin is a widely used chemotherapy drug that targets duplex DNA and was recently shown to react also with G-quadruplex, implying that cisplatin actually may also target G-quadruplex. In this work, we employed magnetic tweezers to investigate the influence of cisplatin on the folding kinetics of single human telomeric G-quadruplex. It was revealed that cisplatin and G-quadruplex interact in two different and competitive ways that depend on cisplatin concentration.

9.
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
10.
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
11.
J Am Chem Soc ; 135(17): 6423-6, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23631592

RESUMO

Single-stranded guanine-rich sequences fold into compact G-quadruplexes. Although G-triplexes have been proposed and demonstrated as intermediates in the folding of G-quadruplexes, there is still a debate on their folding pathways. In this work, we employed magnetic tweezers to investigate the folding kinetics of single human telomeric G-quadruplexes in 100 mM Na(+) buffer. The results are consistent with a model in which the G-triplex is an in-pathway intermediate in the folding of the G-quadruplex. By finely tuning the force exerted on the G-quadruplex, we observed reversible transitions from the G-quadruplex to the G-triplex as well as from the G-triplex to the unfolded coil when the force was increased from 26 to 39 pN. The energy landscape derived from the probability distribution shows clearly that the G-quadruplex goes through an intermediate when it is unfolded, and vice versa.


Assuntos
Quadruplex G , Guanina/química , Telômero/química , Tampões (Química) , Dicroísmo Circular , DNA/química , Campos Eletromagnéticos , Transferência de Energia , Humanos , Cinética , Conformação de Ácido Nucleico , Termodinâmica
12.
Phys Rev E Stat Nonlin Soft Matter Phys ; 79(5 Pt 1): 051912, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19518485

RESUMO

We report observations of in vitro DNA compaction into toroids in the absence of any condensing agent. The DNA toroid formation is induced by geometry confinement from microdroplets on mica surfaces. With AFM imaging we show that the confined DNA molecules may take the form of random coils or semiordered folded loops with large microdroplets, while they readily take the form of compact and ordered toroids when the microdroplet sizes are small enough. To better understand these phenomena, we carried out coarse-grained Brownian dynamics simulation, obtaining results that were in good agreement with the experimental observations. The simulation reveals that the toroid formation is sensitive to not only the microdroplet size, but also the DNA stiffness.


Assuntos
Silicatos de Alumínio/química , DNA/química , DNA/ultraestrutura , Modelos Químicos , Modelos Moleculares , Simulação por Computador , Conformação de Ácido Nucleico , Soluções , Propriedades de Superfície
13.
Langmuir ; 25(10): 5609-13, 2009 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-19260728

RESUMO

We report on the fabrication of a subnanometer-thick epitaxial PbS film on polydiacetylene (PDA) substrate. The film is formed by cation-absorption in PbCl2 solution followed by sulfidation in mixed H2S/N2 gas, exhibiting a flat and smooth surface morphology. The strong adsorbate-surface interaction between the lead hydrolysis complexes and the carboxyl headgroups on the surface of PDA accounts for such a growth mode. We further demonstrate that such an ultrathin film can be used as a buffer layer for both homo- and heteroepitaxial growth of sulfide films. The X-ray and electron diffraction results reveal that the (001) planes of PbS are parallel to the surface of PDA and the (110) planes of PbS are parallel to the (110) planes of PDA.

14.
Nucleic Acids Res ; 37(5): 1400-10, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19129234

RESUMO

Structural properties of single lambda DNA treated with anti-cancer drug cisplatin were studied with magnetic tweezers and AFM. Under the effect of low-concentration cisplatin, the DNA became more flexible, with the persistence length decreased significantly from approximately 52 to 15 nm. At a high drug concentration, a DNA condensation phenomenon was observed. Based on experimental results from both single-molecule and AFM studies, we propose a model to explain this kind of DNA condensation by cisplatin: first, di-adducts induce local distortions of DNA. Next, micro-loops of approximately 20 nm appear through distant crosslinks. Then, large aggregates are formed through further crosslinks. Finally, DNA is condensed into a compact globule. Experiments with Pt(dach)Cl(2) indicate that oxaliplatin may modify the DNA structures in the same way as cisplatin. The observed loop structure formation of DNA may be an important feature of the effect of platinum anti-cancer drugs that are analogous to cisplatin in structure.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , DNA/efeitos dos fármacos , DNA/ultraestrutura , Bacteriófago lambda/genética , DNA/química , DNA Viral/química , DNA Viral/efeitos dos fármacos , DNA Viral/ultraestrutura , Elasticidade , Microscopia de Força Atômica , Conformação de Ácido Nucleico , Compostos Organoplatínicos/farmacologia , Oxaliplatina
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