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1.
Nat Commun ; 10(1): 2159, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089141

RESUMO

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Drosophila/metabolismo , Modelos Moleculares , DNA Helicases/isolamento & purificação , Proteínas de Drosophila/isolamento & purificação , Fenômenos Magnéticos , Pinças Ópticas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/métodos
2.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29976672

RESUMO

Herpes simplex virus 1 (HSV-1) UL51 is a phosphoprotein that functions in the final envelopment in the cytoplasm and viral cell-cell spread, leading to efficient viral replication in cell cultures. To clarify the mechanism by which UL51 is regulated in HSV-1-infected cells, we focused on the phosphorylation of UL51. Mass spectrometry analysis of purified UL51 identified five phosphorylation sites in UL51. Alanine replacement of one of the identified phosphorylation sites in UL51, serine 184 (Ser-184), but not the other identified phosphorylation sites, significantly reduced viral replication and cell-cell spread in HaCaT cells. This mutation induced membranous invaginations adjacent to the nuclear membrane, the accumulation of primary enveloped virions in the invaginations and perinuclear space, and mislocalized UL34 and UL31 in punctate structures at the nuclear membrane; however, it had no effect on final envelopment in the cytoplasm of HaCaT cells. Of note, the alanine mutation in UL51 Ser-184 significantly reduced the mortality of mice following ocular infection. Phosphomimetic mutation in UL51 Ser-184 partly restored the wild-type phenotype in cell cultures and in mice. Based on these results, we concluded that some UL51 functions are specifically regulated by phosphorylation at Ser-184 and that this regulation is critical for HSV-1 replication in cell cultures and pathogenicity in vivoIMPORTANCE HSV-1 UL51 is conserved in all members of the Herpesviridae family. This viral protein is phosphorylated and functions in viral cell-cell spread and cytoplasmic virion maturation in HSV-1-infected cells. Although the downstream effects of HSV-1 UL51 have been clarified, there is a lack of information on how this viral protein is regulated as well as the significance of the phosphorylation of this protein in HSV-1-infected cells. In this study, we show that the phosphorylation of UL51 at Ser-184 promotes viral replication, cell-cell spread, and nuclear egress in cell cultures and viral pathogenicity in mice. This is the first report to identify the mechanism by which UL51 is regulated as well as the significance of UL51 phosphorylation in HSV-1 infection. Our study may provide insights into the regulatory mechanisms of other herpesviral UL51 homologs.


Assuntos
DNA Helicases/química , DNA Helicases/fisiologia , DNA Primase/química , DNA Primase/fisiologia , Herpesvirus Humano 1/patogenicidade , Proteínas Virais/química , Proteínas Virais/fisiologia , Liberação de Vírus , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Cercopithecus aethiops , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DNA Primase/genética , DNA Primase/isolamento & purificação , Olho/virologia , Células HEK293 , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Humanos , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases , Células Vero , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Vírion/fisiologia , Virulência , Montagem de Vírus
3.
J Biol Chem ; 292(28): 11927-11936, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28533432

RESUMO

Proper chromatin regulation is central to genome function and maintenance. The group III chromodomain-helicase-DNA-binding (CHD) family of ATP-dependent chromatin remodeling enzymes, comprising CHD6, CHD7, CHD8, and CHD9, has well-documented roles in transcription regulation, impacting both organism development and disease etiology. These four enzymes are similar in their constituent domains, but they fill surprisingly non-redundant roles in the cell, with deficiencies in individual enzymes leading to dissimilar disease states such as CHARGE syndrome or autism spectrum disorders. The mechanisms explaining their divergent, non-overlapping functions are unclear. In this study, we performed an in-depth biochemical analysis of purified CHD6, CHD7, and CHD8 and discovered distinct differences in chromatin remodeling specificities and activities among them. We report that CHD6 and CHD7 both bind with high affinity to short linker DNA, whereas CHD8 requires longer DNA for binding. As a result, CHD8 slides nucleosomes into positions with more flanking linker DNA than CHD7. Moreover, we found that, although CHD7 and CHD8 slide nucleosomes, CHD6 disrupts nucleosomes in a distinct non-sliding manner. The different activities of these enzymes likely lead to differences in chromatin structure and, thereby, transcriptional control, at the enhancer and promoter loci where these enzymes bind. Overall, our work provides a mechanistic basis for both the non-redundant roles and the diverse mutant disease states of these enzymes in vivo.


Assuntos
Trifosfato de Adenosina/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nucleossomos/enzimologia , Fatores de Transcrição/metabolismo , Animais , Transporte Biológico , DNA/química , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DNA Recombinante/química , DNA Recombinante/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Células HeLa , Humanos , Hidrólise , Cinética , Peso Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Nucleossomos/metabolismo , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação
4.
Methods Enzymol ; 582: 121-136, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28062032

RESUMO

Helicases control the accessibility of single-stranded (ss) nucleic acid (NA) generated as a transient intermediate during almost every step in cells related to nucleic acid metabolisms. For subsequent processing, however, helicases need to adjust the pace of unwinding adequately to avoid ssNA exposure to nucleases. Therefore, understanding how the unwinding process of helicases is regulated is crucial to address genome integrity and repair mechanisms. Using single-molecule fluorescence-force spectroscopy with fluorescence localization, we recently observed the stoichiometry of UvrD helicase, which determines the functions of UvrD: translocation and unwinding. For the first time, we provide direct evidence that a UvrD dimer is required to initiate the unwinding pathway. Moreover, with subpixel precision of fluorescence localization, the dynamic parameters of helicases can be obtained directly. Here, we present detailed single-molecule assays for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein-nucleic acid interactions. These single-molecule approaches are generally applicable to many other protein-nucleic acid systems.


Assuntos
DNA Helicases/isolamento & purificação , DNA de Cadeia Simples/isolamento & purificação , Pinças Ópticas , Imagem Individual de Molécula/métodos , DNA Helicases/química , DNA de Cadeia Simples/química , Escherichia coli/química
5.
Methods Enzymol ; 582: 137-169, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28062033

RESUMO

We describe the design, construction, and application of an instrument combining dual-trap, high-resolution optical tweezers and a confocal microscope. This hybrid instrument allows nanomechanical manipulation and measurement simultaneously with single-molecule fluorescence detection. We present the general design principles that overcome the challenges of maximizing optical trap resolution while maintaining single-molecule fluorescence sensitivity, and provide details on the construction and alignment of the instrument. This powerful new tool is just beginning to be applied to biological problems. We present step-by-step instructions on an application of this technique that highlights the instrument's capabilities, detecting conformational dynamics in a nucleic acid-processing enzyme.


Assuntos
DNA Helicases/isolamento & purificação , Microscopia Confocal/métodos , Pinças Ópticas , Imagem Individual de Molécula/métodos , DNA Helicases/química , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos
6.
Methods Enzymol ; 582: 387-414, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28062043

RESUMO

Nanopores are emerging as new single-molecule tools in the study of enzymes. Based on the progress in nanopore sequencing of DNA, a tool called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) was developed to measure the movement of enzymes along DNA in real time. In this new method, an enzyme is loaded onto a DNA (or RNA) molecule. A single-stranded DNA end of this complex is drawn into a nanopore by an electrostatic potential that is applied across the pore. The single-stranded DNA passes through the pore's constriction until the enzyme comes into contact with the pore. Further progression of the DNA through the pore is then controlled by the enzyme. An ion current that flows through the pore's constriction is modulated by the DNA in the constriction. Analysis of ion current changes reveals the advance of the DNA with high spatiotemporal precision, thereby providing a real-time record of the enzyme's activity. Using an engineered version of the protein nanopore MspA, SPRNT has spatial resolution as small as 40pm at millisecond timescales, while simultaneously providing the DNA's sequence within the enzyme. In this chapter, SPRNT is introduced and its extraordinary potential is exemplified using the helicase Hel308. Two distinct substates are observed for each one-nucleotide advance; one of these about half-nucleotide long steps is ATP dependent and the other is ATP independent. The spatiotemporal resolution of this low-cost single-molecule technique lifts the study of enzymes to a new level of precision, enabling exploration of hitherto unobservable enzyme dynamics in real time.


Assuntos
DNA Helicases/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Nanoporos , Imagem Individual de Molécula/métodos , DNA Helicases/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Nucleotídeos , Análise de Sequência de DNA/métodos
7.
Malar J ; 15(1): 526, 2016 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-27809838

RESUMO

BACKGROUND: Malaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. Thus, identifying new drug targets is urgently needed. Helicases play key roles in a wide range of cellular activities involving DNA and RNA transactions, making them attractive anti-malarial drug targets. METHODS: ATP-dependent DNA helicase gene (PfRuvB3) of Plasmodium falciparum strain K1, a chloroquine and pyrimethamine-resistant strain, was inserted into pQE-TriSystem His-Strep 2 vector, heterologously expressed and affinity purified. Identity of recombinant PfRuvB3 was confirmed by western blotting coupled with tandem mass spectrometry. Helicase and ATPase activities were characterized as well as co-factors required for optimal function. RESULTS: Recombinant PfRuvB3 has molecular size of 59 kDa, showing both DNA helicase and ATPase activities. Its helicase activity is dependent on divalent cations (Cu2+, Mg2+, Ni+2 or Zn+2) and ATP or dATP but is inhibited by high NaCl concentration (>100 mM). PfPuvB3 is unable to act on blunt-ended duplex DNA, but manifests ATPase activity in the presence of either single- or double-stranded DNA. PfRuvB3.is inhibited by doxorubicin, daunorubicin and netropsin, known DNA helicase inhibitors. CONCLUSIONS: Purified recombinant PfRuvB3 contains both DNA helicase and ATPase activities. Differences in properties of RuvB between the malaria parasite obtained from the study and human host provide an avenue leading to the development of novel drugs targeting specifically the malaria form of RuvB family of DNA helicases.


Assuntos
DNA Helicases/metabolismo , Plasmodium falciparum/enzimologia , Proteínas Recombinantes/metabolismo , Western Blotting , Cátions Bivalentes/metabolismo , Clonagem Molecular , Coenzimas/análise , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/isolamento & purificação , Inibidores Enzimáticos/análise , Expressão Gênica , Metais/metabolismo , Peso Molecular , Plasmodium falciparum/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Espectrometria de Massas em Tandem
8.
Methods Enzymol ; 581: 1-32, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27793277

RESUMO

The unique translocation and DNA unwinding properties of DNA helicases can be concealed by the stochastic behavior of enzyme molecules within the necessarily large populations used in ensemble experiments. With recent technological advances, the direct visualization of helicases acting on individual DNA molecules has contributed significantly to the current understanding of their mechanisms of action and biological functions. The combination of single-molecule techniques that enable both manipulation of individual protein or DNA molecules and visualization of their actions has made it possible to literally see novel and unique biochemical characteristics that were previously masked. Here, we describe the execution and use of single-molecule fluorescence imaging techniques, focusing on methods that include optical trapping in conjunction with epifluorescent imaging, and also surface immobilization in conjunction with total internal reflection fluorescence visualization. Combined with microchannel flow cells and microfluidic control, these methods allow individual fluorescently labeled protein and DNA molecules to be imaged and tracked, affording measurement of DNA unwinding and translocation at single-molecule resolution.


Assuntos
Bioquímica/métodos , DNA Helicases/isolamento & purificação , Imagem Molecular/métodos , DNA Helicases/genética , Microscopia de Fluorescência , Pinças Ópticas
9.
Methods ; 108: 1-3, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27565743

RESUMO

In this special Methods collection on DNA helicases, I have solicited articles from leading experts in the field with a priority to gather a defined series of papers on highly relevant topics that encompass biological, biochemical, and biophysical aspects of helicase function. The experimental approaches described provide an opportunity for both new and more experienced scientists to use the information for the design of their own investigations. The reader will find detailed methods for single-molecule studies, novel biochemical experiments, genetic analyses, and cell biological assays in a variety of systems with an emphasis placed on state-of-the-art techniques to measure helicase function. Contributing authors were strongly encouraged to provide a carefully constructed description of the methods employed so that others might use this information in a manner that will be useful for their own particular application and helicase of interest. This special issue of Methods dedicated to DNA helicases offers readers a treasure chest of unique experimental approaches and protocols focused on rapidly developing techniques that are useful for studying both in vivo and in vitro aspects of helicase function.


Assuntos
DNA Helicases/química , DNA Helicases/genética , DNA/genética , DNA/química , DNA Helicases/isolamento & purificação
10.
Methods ; 108: 14-23, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27223403

RESUMO

DNA helicases participate in virtually all aspects of cellular DNA metabolism by using ATP-fueled directional translocation along the DNA molecule to unwind DNA duplexes, dismantle nucleoprotein complexes, and remove non-canonical DNA structures. Post-translational modifications and helicase interacting partners are often viewed as determining factors in controlling the switch between bona fide helicase activity and other functions of the enzyme that do not involve duplex separation. The bottleneck in developing a mechanistic understanding of human helicases and their control by post-translational modifications is obtaining sufficient quantities of the modified helicase for traditional structure-functional analyses and biochemical reconstitutions. This limitation can be overcome by single-molecule analysis, where several hundred surface-tethered molecules are sufficient to obtain a complete kinetic and thermodynamic description of the helicase-mediated substrate binding and rearrangement. Synthetic oligonucleotides site-specifically labeled with Cy3 and Cy5 fluorophores can be used to create a variety of DNA substrates that can be used to characterize DNA binding, as well as helicase translocation and duplex unwinding activities. This chapter describes "single-molecule sorting", a robust experimental approach to simultaneously quantify, and distinguish the activities of helicases carrying their native post-translational modifications. Using this technique, a DNA helicase of interest can be produced and biotinylated in human cells to enable surface-tethering for the single-molecule studies by total internal reflection fluorescence microscopy. The pool of helicases extracted from the cells is expected to contain a mixture of post-translationally modified and unmodified enzymes, and the contributions from either population can be monitored separately, but in the same experiment providing a direct route to evaluating the effect of a given modification.


Assuntos
DNA Helicases/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Citometria de Fluxo/métodos , Imagem Individual de Molécula/métodos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , DNA/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética
11.
Methods ; 105: 90-8, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27038745

RESUMO

Recent advances in high-throughput single-molecule magnetic tweezers have paved the way for obtaining information on individual molecules as well as ensemble-averaged behavior in a single assay. Here we describe how to design robust high-throughput magnetic tweezers assays that specifically require application of high forces (>20pN) for prolonged periods of time (>1000s). We elaborate on the strengths and limitations of the typical construct types that can be used and provide a step-by-step guide towards a high tether yield assay based on two examples. Firstly, we discuss a DNA hairpin assay where force-induced strand separation triggers a tight interaction between DNA-binding protein Tus and its binding site Ter, where forces up to 90pN for hundreds of seconds were required to dissociate Tus from Ter. Secondly, we show how the LTag helicase of Simian virus 40 unwinds dsDNA, where a load of 36pN optimizes the assay readout. The approaches detailed here provide guidelines for the high-throughput, quantitative study of a wide range of DNA-protein interactions.


Assuntos
DNA Helicases/química , Proteínas de Ligação a DNA/química , Ensaios de Triagem em Larga Escala/métodos , Imagem Individual de Molécula/métodos , DNA/química , DNA Helicases/isolamento & purificação , Proteínas de Ligação a DNA/genética , Pinças Ópticas , Vírus 40 dos Símios/enzimologia
12.
Nucleic Acids Res ; 43(18): 8942-54, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26384418

RESUMO

ScPif1 DNA helicase is the prototypical member of a 5'-to-3' helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5'-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA-RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , DNA Helicases/metabolismo , DNA/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , DNA/química , DNA Helicases/química , DNA Helicases/isolamento & purificação , Quadruplex G , Especificidade por Substrato
13.
PLoS One ; 9(11): e113293, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25409515

RESUMO

8,5' cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair.


Assuntos
DNA Helicases/metabolismo , Desoxiadenosinas/farmacologia , Desoxiguanosina/farmacologia , Archaea/enzimologia , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , DNA/química , Dano ao DNA , DNA Helicases/isolamento & purificação , Reparo do DNA , Desoxiadenosinas/síntese química , Desoxiguanosina/síntese química , Humanos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
14.
J Vis Exp ; (92): e51720, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25407439

RESUMO

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex's chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.


Assuntos
DNA Helicases/isolamento & purificação , Cromatografia de Afinidade/métodos , DNA Helicases/biossíntese , DNA Helicases/genética , DNA Helicases/imunologia , DNA Complementar/genética , Epitopos/química , Epitopos/imunologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos
15.
Cell ; 159(4): 869-83, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25417162

RESUMO

X chromosome inactivation (XCI) depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). PRC2 is also targeted to other sites throughout the genome to effect transcriptional repression. Using XCI as a model, we apply an unbiased proteomics approach to isolate Xist and PRC2 regulators and identified ATRX. ATRX unexpectedly functions as a high-affinity RNA-binding protein that directly interacts with RepA/Xist RNA to promote loading of PRC2 in vivo. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X chromosome. Moreover, epigenomic profiling reveals that genome-wide targeting of PRC2 depends on ATRX, as loss of ATRX leads to spatial redistribution of PRC2 and derepression of Polycomb responsive genes. Thus, ATRX is a required specificity determinant for PRC2 targeting and function.


Assuntos
DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/metabolismo , Inativação do Cromossomo X , Animais , DNA Helicases/isolamento & purificação , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Proteínas Nucleares/isolamento & purificação , Proteína Nuclear Ligada ao X
16.
BMC Plant Biol ; 14: 283, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25311683

RESUMO

BACKGROUND: Helicases play crucial role in almost all the nucleic acid metabolism including replication, repair, recombination, transcription, translation, ribosome biogenesis and splicing and these processes regulate plant growth and development. It is suggested that helicases play essential roles in stabilizing growth in plants under stress because their presence in the stress-induced ORFs has been identified. Moreover in a recent study we have reported that SUV3 helicase from Oryza sativa (OsSUV3) functions in salinity stress tolerance in transgenic rice by improving the antioxidant machinery. SUV3 helicase has been identified and characterized from yeast and human systems but the properties and functions of plant SUV3 are poorly understood. RESULTS: In this study, the purification and extensive characterization of recombinant OsSUV3 protein (67 kDa) is presented. OsSUV3 binds to DNA and RNA and exhibits DNA as well as RNA-dependent ATPase activities. It also contains the characteristic DNA and RNA helicase activity. OsSUV3 can use mainly ATP or dATP as energy source for the unwinding activity and it cannot unwind the blunt-end duplex DNA substrate. It is interesting to note that OsSUV3 unwinds DNA in both the 5'-3' and 3'-5 directions and thus its activity is bipolar in vitro. The Km values of OsSUV3 are 0.51 nM and 0.95 nM for DNA helicase and RNA helicase, respectively. CONCLUSIONS: This study is the first direct evidence to show the bipolar DNA helicase activity of OsSUV3 protein. The unique properties of OsSUV3 including its dual helicase activity imply that it could be a multifunctional protein involved in biologically significant process of DNA and RNA metabolisms. These results should make significant contribution towards better understanding of SUV3 protein in plants.


Assuntos
DNA Helicases/metabolismo , Oryza/enzimologia , RNA Helicases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Helicases/genética , DNA Helicases/isolamento & purificação , Nucleotídeos de Desoxiadenina/metabolismo , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Ligação Proteica , RNA Helicases/genética , RNA Helicases/isolamento & purificação , Proteínas Recombinantes , Salinidade
17.
Proc Natl Acad Sci U S A ; 111(43): 15390-5, 2014 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-25313033

RESUMO

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.


Assuntos
DNA Polimerase II/metabolismo , Replicação do DNA , Holoenzimas/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Cromatografia em Gel , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , DNA Circular/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Fatores de Tempo
18.
Plant Mol Biol ; 85(6): 639-51, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24908423

RESUMO

DEAD-box helicases play essential role in DNA and RNA metabolism such as replication, repair, recombination, transcription, translation, ribosome biogenesis and splicing which regulate plant growth and development. The presence of helicases in the stress-induced ORFs identified by cDNA microarray indicates that helicases might be playing an important role in stabilizing growth in plants under stress. p68 DEAD-box helicase has been identified and characterized from animal systems but the properties and functions of plant p68 are poorly understood. In this study, the identification, purification and characterization of recombinant p68 from Pisum sativum (Psp68) is presented. Psp68 possesses all the characteristic motifs like DEAD-box ATP-binding and helicase C terminal motifs and is structurally similar to human p68 homologue. Psp68 exhibits ATPase activity in the presence of both DNA and RNA and it binds to DNA as well as RNA. It contains the characteristic RNA helicase activity. Interestingly Psp68 also shows the unique DNA helicase activity, which is bipolar in nature (unwinds DNA in both the 5'-3' and 3'-5' directions). The Km values of Psp68 for ATPase are 0.5126 and 0.9142 mM in the presence of DNA and RNA, respectively. The Km values of Psp68 are 1.6129 and 1.14 nM for DNA helicase and RNA helicase, respectively. The unique properties of Psp68 suggest that it could be a multifunctional protein involved in different aspect of DNA and RNA metabolism. This discovery should make an important contribution to better understanding of nucleic acids metabolism plants.


Assuntos
RNA Helicases DEAD-box/fisiologia , DNA Helicases/fisiologia , Ervilhas/enzimologia , Proteínas de Plantas/fisiologia , Sequência de Aminoácidos , Clonagem Molecular , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/isolamento & purificação , DNA Helicases/química , DNA Helicases/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
19.
Epigenetics ; 9(5): 693-7, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24565939

RESUMO

The HepA-related protein (HARP/SMARCAL1) is an ATP-dependent annealing helicase that is capable of rewinding DNA structures that are stably unwound due to binding of the single-stranded DNA (ssDNA)-binding protein Replication Protein A (RPA). HARP has been implicated in maintaining genome integrity through its role in DNA replication and repair, two processes that generate RPA-coated ssDNA. In addition, mutations in HARP cause a rare disease known as Schimke immuno-osseous dysplasia. In this study, we purified HARP containing complexes with the goal of identifying the predominant factors that stably associate with HARP. We found that HARP preferentially interacts with RPA molecules that are bound to the DNA-dependent protein kinase (DNA-PK). We also found that RPA is phosphorylated by DNA-PK in vitro, while the RPA-HARP complexes are not. Our results suggest that, in addition to its annealing helicase activity, which eliminates the natural binding substrate for RPA, HARP blocks the phosphorylation of RPA by DNA-PK.


Assuntos
DNA Helicases/isolamento & purificação , Proteína Quinase Ativada por DNA/isolamento & purificação , Proteínas Nucleares/isolamento & purificação , Proteína de Replicação A/isolamento & purificação , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fosforilação , Proteína de Replicação A/metabolismo
20.
J Virol Methods ; 194(1-2): 33-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23948158

RESUMO

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that is prevalent in south-east Asia. Because there is no specific antiviral agent, JEV still causes a high rate of neurologic sequelae and mortality in humans. The helicase encoded by the NS3 gene of JEV has emerged recently as a novel antiviral target for treatment. In this study, a soluble recombinant JEV helicase protein was expressed and purified. Methods for detecting the ATP hydrolysis and nucleic acid unwinding activity were developed by luminescence and fluorescence resonance energy transfer (FRET). The concentrations of enzyme, substrate, capture strand, ATP, and divalent ions were optimised in the ATPase and helicase reactions. The feasibility of using these two methods for high-throughput screening of NS3 helicase inhibitors is discussed.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Helicases/análise , Vírus da Encefalite Japonesa (Espécie)/enzimologia , Ácidos Nucleicos/metabolismo , Proteínas não Estruturais Virais/análise , DNA Helicases/genética , DNA Helicases/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Medições Luminescentes , RNA Helicases/análise , RNA Helicases/genética , RNA Helicases/isolamento & purificação , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Serina Endopeptidases/análise , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação
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