RESUMEN
Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS-TIPIN (respectively known as Ctf4, Mrc1 and Tof1-Csm3 in Saccharomyces cerevisiae) are organized around the CDC45-MCM-GINS (CMG) replicative helicase1-7. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18-RFC. We show how CLASPIN and TIMELESS-TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome8, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα9,10, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN , Complejos Multienzimáticos , ADN/biosíntesis , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Humanos , Complejos Multienzimáticos/química , Complejos Multienzimáticos/aislamiento & purificación , Factores de TiempoRESUMEN
Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions.
Asunto(s)
ADN Helicasas/genética , ARN Helicasas/genética , Thermococcales/enzimología , Adenosina Trifosfatasas/genética , Proteínas Arqueales/química , ADN/química , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Filogenia , ARN/química , ARN Helicasas/aislamiento & purificación , ARN Helicasas/metabolismo , Homología de Secuencia de Aminoácido , Thermococcales/genética , Thermococcales/metabolismoRESUMEN
Liquid-liquid phase separation (LLPS) is a mechanism of intracellular organization that underlies the assembly of a variety of RNP granules. Fundamental biophysical principles governing LLPS during granule assembly have been revealed by simple in vitro systems, but these systems have limitations when studying the biology of complex, multicomponent RNP granules. Visualization of RNP granules in cells has validated key principles revealed by simple in vitro systems, but this approach presents difficulties for interrogating biophysical features of RNP granules and provides limited ability to manipulate protein, nucleic acid, or small molecule concentrations. Here, we introduce a system that builds upon recent insights into the mechanisms underlying RNP granule assembly and permits high-fidelity reconstitution of stress granules and the granular component of nucleoli in mammalian cellular lysate. This system fills the gap between simple in vitro systems and live cells and allows for a variety of studies of membraneless organelles, including the development of therapeutics that modify properties of specific condensates.
Asunto(s)
Nucléolo Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , Mamíferos/metabolismo , Estrés Fisiológico , Animales , Extractos Celulares , Línea Celular , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas de Unión a Poli-ADP-Ribosa/aislamiento & purificación , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN/metabolismo , ARN Helicasas/aislamiento & purificación , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/aislamiento & purificación , Proteínas con Motivos de Reconocimiento de ARN/metabolismoRESUMEN
The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.
Asunto(s)
ADN Helicasas/genética , Reparación del ADN , Neoplasias/genética , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Secuencia Conservada/genética , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Células HEK293 , Humanos , Mutación , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Unión Proteica/genética , Conformación Proteica en Hélice alfa/genética , Dominios Proteicos/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/metabolismoRESUMEN
DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of â¼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa I/metabolismo , Geobacillus stearothermophilus/enzimología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , ADN , ADN Helicasas/química , ADN Helicasas/aislamiento & purificación , Desoxirribonucleasa I/química , Desoxirribonucleasa I/aislamiento & purificaciónRESUMEN
BRG1/SMARCA4 and its paralog BRM/SMARCA2 are the ATPase subunits of human SWI/SNF chromatin remodeling complexes. These multisubunit assemblies can act as either tumor suppressors or drivers of cancer, and inhibiting both BRG1 and BRM, is emerging as an effective therapeutic strategy in diverse cancers. BRG1 and BRM contain a BRK domain. The function of this domain is unknown, but it is often found in proteins involved in transcription and developmental signaling in higher eukaryotes, in particular in proteins that remodel chromatin. We report the NMR structure of the BRG1 BRK domain. It shows similarity to the glycine-tyrosine-phenylalanine (GYF) domain, an established protein-protein interaction module. Computational peptide-binding-site analysis of the BRK domain identifies a binding site that coincides with a highly conserved groove on the surface of the protein. This sets the scene for experiments to elucidate the role of this domain, and evaluate the potential of targeting it for cancer therapy.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN Helicasas/química , Proteínas Nucleares/química , Factores de Transcripción/química , Cromatina/química , Cromatina/metabolismo , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , Humanos , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Unión Proteica , Conformación Proteica , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Dominios Homologos srcRESUMEN
Liver sinusoidal endothelial cells play a key role maintaining the hepatic homeostasis, the disruption of which is associated with such end-stage liver diseases as hepatocellular carcinoma and cirrhosis. In the present study we investigated the role of brahma-related gene 1 (BRG1), a chromatin remodeling protein, in regulating endothelial transcription and the implication in liver fibrosis. We report that endothelial-specific deletion of BRG1 in mice attenuated liver fibrosis induced by injection with thioacetamide (TAA). Coincidently, alleviation of liver fibrosis as a result of endothelial BRG1 deletion was accompanied by an up-regulation of eNOS activity and NO bioavailability. In cultured endothelial cells, exposure to lipopolysaccharide (LPS) suppressed eNOS activity whereas BRG1 depletion with small interfering RNA restored eNOS-dependent NO production. Further analysis revealed that BRG1 was recruited to the caveolin-1 (CAV1) promoter by Sp1 and activated transcription of CAV1, which in turn inhibited eNOS activity. Mechanistically, BRG1 interacted with the H3K4 trimethyltransferase MLL1 to modulate H3K4 trimethylation surrounding the CAV1 promoter thereby contributing to LPS-induced CAV1 activation. In conclusion, our data unveil a novel role for BRG1 in the regulation of endothelial function and liver fibrosis.
Asunto(s)
ADN Helicasas/metabolismo , Células Endoteliales/metabolismo , Fibrosis/metabolismo , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , ADN Helicasas/deficiencia , ADN Helicasas/aislamiento & purificación , Fibrosis/inducido químicamente , Humanos , Hígado/efectos de los fármacos , Ratones , Óxido Nítrico/análisis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/aislamiento & purificación , Tioacetamida , Factores de Transcripción/deficiencia , Factores de Transcripción/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN , Proteínas de Drosophila/metabolismo , Modelos Moleculares , ADN Helicasas/aislamiento & purificación , Proteínas de Drosophila/aislamiento & purificación , Fenómenos Magnéticos , Pinzas Ópticas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
DNA double-strand breaks (DSBs) are a potentially lethal DNA lesions that disrupt both the physical and genetic continuity of the DNA duplex. Homologous recombination (HR) is a universally conserved genome maintenance pathway that initiates via nucleolytic processing of the broken DNA ends (resection). Eukaryotic DNA resection is catalyzed by the resectosome-a multicomponent molecular machine consisting of the nucleases DNA2 or Exonuclease 1 (EXO1), Bloom's helicase (BLM), the MRE11-RAD50-NBS1 (MRN) complex, and additional regulatory factors. Here, we describe methods for purification and single-molecule imaging and analysis of EXO1, DNA2, and BLM. We also describe how to adapt resection assays to the high-throughput single-molecule DNA curtain assay. By organizing hundreds of individual molecules on the surface of a microfluidic flowcell, DNA curtains visualize protein complexes with the required spatial and temporal resolution to resolve the molecular choreography during critical DNA-processing reactions.
Asunto(s)
Técnicas Analíticas Microfluídicas/métodos , Reparación del ADN por Recombinación , Imagen Individual de Molécula/métodos , Roturas del ADN de Doble Cadena , ADN Helicasas/análisis , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , Enzimas Reparadoras del ADN/análisis , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/aislamiento & purificación , Exodesoxirribonucleasas/análisis , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Microscopía Fluorescente/métodos , Puntos Cuánticos/química , RecQ Helicasas/genética , RecQ Helicasas/aislamiento & purificación , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Efficient replication and repair of the genome requires a multitude of protein-DNA transactions. These interactions can result in a variety of consequences for DNA such as the unwinding of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), the annealing of complementary ssDNAs, or the exchange of ssDNA with one strand of a dsDNA duplex. Some DNA helicases possess all three activities, but many DNA-interacting proteins can also catalyze one or more of these reactions. Assays that quantify these activities are an important first step in characterizing these protein-DNA interactions in vitro. Here, we describe methods for the formation of dsDNA substrates and the assays that can be used to biochemically characterize proteins that can unwind, anneal, and/or exchange DNA strands.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Pruebas de Enzimas/métodos , ADN Helicasas/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Marcaje Isotópico/métodos , Electroforesis en Gel de Poliacrilamida Nativa/métodos , Radioisótopos de Fósforo/química , Unión ProteicaRESUMEN
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.
Asunto(s)
ADN Helicasas/química , ADN Helicasas/fisiología , ADN Primasa/química , ADN Primasa/fisiología , Herpesvirus Humano 1/patogenicidad , Proteínas Virales/química , Proteínas Virales/fisiología , Liberación del Virus , Replicación Viral , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Chlorocebus aethiops , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , ADN Primasa/genética , ADN Primasa/aislamiento & purificación , Ojo/virología , Células HEK293 , Herpes Simple/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Humanos , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas , Células Vero , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Virión/fisiología , Virulencia , Ensamble de VirusRESUMEN
Accurate repair of DNA double-strand breaks (DSBs) is carried out by homologous recombination. In order to repair DNA breaks by the recombination pathway, the 5'-terminated DNA strand at DSB sites must be first nucleolytically processed to produce 3'-overhang. The process is termed DNA end resection and involves the interplay of several nuclease complexes. DNA end resection commits DSB repair to the recombination pathway including a process termed single-strand annealing, as resected DNA ends are generally nonligatable by the competing nonhomologous end-joining machinery. Biochemical reconstitution experiments provided invaluable mechanistic insights into the DNA end resection pathways. In this chapter, we describe preparation procedures of key proteins involved in DNA end resection in human cells, including the MRE11-RAD50-NBS1 complex, phosphorylated variant of CtIP, the DNA2 nuclease-helicase with its helicase partners Bloom (BLM) or Werner (WRN), as well as the single-stranded DNA-binding protein replication protein A. The availability of recombinant DNA end resection factors will help to further elucidate resection mechanisms and regulatory processes that may involve novel protein partners and posttranslational modifications.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Roturas del ADN de Doble Cadena , Pruebas de Enzimas/métodos , Proteínas Recombinantes/aislamiento & purificación , Reparación del ADN por Recombinación , Ácido Anhídrido Hidrolasas , Animales , Baculoviridae/genética , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/aislamiento & purificación , Enzimas Reparadoras del ADN/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas , Pruebas de Enzimas/instrumentación , Humanos , Proteína Homóloga de MRE11/aislamiento & purificación , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , RecQ Helicasas/aislamiento & purificación , RecQ Helicasas/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicación A/aislamiento & purificación , Proteína de Replicación A/metabolismo , Células Sf9 , Spodoptera , Transfección/métodos , Helicasa del Síndrome de Werner/aislamiento & purificación , Helicasa del Síndrome de Werner/metabolismoRESUMEN
Budding yeast Dmc1 is a member of the RecA family of strand exchange proteins essential for homologous recombination (HR) during meiosis. Dmc1 mediates the steps of homology search and DNA strand exchange reactions that are central to HR. To achieve optimum activity, Dmc1 requires a number of accessory factors. Although methods for purification of Dmc1 and many of its associated factors have been described (Binz, Dickson, Haring, & Wold, 2006; Busygina et al., 2013; Chan, Brown, Qin, Handa, & Bishop, 2014; Chi et al., 2006; Cloud, Chan, Grubb, Budke, & Bishop, 2012; Nimonkar, Amitani, Baskin, & Kowalczykowski, 2007; Van Komen, Macris, Sehorn, & Sung, 2006), Dmc1 has been particularly difficult to purify because of its tendency to aggregate. Here, we provide an alternative and simple high-yield purification method for recombinant Dmc1 that is active and responsive to stimulation by accessory factors. The same method may be used for purification of recombinant Rdh54 (a.k.a. Tid1) and other HR proteins with minor adjustments. We also describe an economical and sensitive D-loop assay for strand exchange proteins that uses fluorescent dye-tagged, rather than radioactive, ssDNA substrates.
Asunto(s)
Proteínas de Ciclo Celular/aislamiento & purificación , ADN Helicasas/aislamiento & purificación , ADN-Topoisomerasas/aislamiento & purificación , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Reparación del ADN por Recombinación , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/instrumentación , Cromatografía por Intercambio Iónico/métodos , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN-Topoisomerasas/química , ADN-Topoisomerasas/metabolismo , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Colorantes Fluorescentes/química , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Helicases are crucial participants in many types of DNA repair reactions, including homologous recombination. The properties of these enzymes can be assayed by traditional bulk biochemical analysis; however, these types of assays cannot directly access some types of information. In particular, bulk biochemical assays cannot readily access information that may be obscured in population averages. Single-molecule assays offer the potential advantage of being able to visualize the molecules in question in real time, thus providing direct access to questions relating to translocation velocity, processivity, and insights into how helicases may behave on different types of substrates. Here, we describe the use of single-stranded DNA (ssDNA) curtains as an assay for directly viewing the behavior of the Saccharomyces cerevisiae Srs2 helicase on single molecules of ssDNA. When used with total internal reflection fluorescence microscopy, these methods can be used to track the binding and movements of individual helicase complexes, and allow new insights into helicase behaviors at the single-molecule level.
Asunto(s)
ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Pruebas de Enzimas/métodos , Reparación del ADN por Recombinación , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagen Individual de Molécula/métodos , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/aislamiento & purificación , Pruebas de Enzimas/instrumentación , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Imagen Individual de Molécula/instrumentación , Coloración y Etiquetado/métodosRESUMEN
DNA end resection initiates the largely accurate repair of DNA double-strand breaks (DSBs) by homologous recombination. Specifically, recombination requires the formation of 3' overhangs at DSB sites, which is carried out by nucleases that specifically degrade 5'-terminated DNA. In most cases, DNA end resection is a two-step process, comprising of initial short-range followed by more processive long-range resection. In this chapter, we describe selected assays that reconstitute both the short- and long-range pathways. First, we define methods to study the exonuclease and endonuclease activities of the MRE11-RAD50-NBS1 (MRN) complex in conjunction with phosphorylated cofactor CtIP. This reaction is particularly important to initiate processing of DNA breaks and to recruit components belonging to the subsequent long-range pathway. Next, we describe assays that reconstitute the concerted reactions of Bloom (BLM) or Werner (WRN) helicases that function together with the DNA2 nuclease-helicase, and which are as a complex capable to resect DNA of kilobases in length. The reconstituted reactions allow us to understand how the resection pathways function at the molecular level. The assays will be invaluable to define regulatory mechanisms and to identify inhibitory compounds, which may be valuable in cancer therapy.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Roturas del ADN de Doble Cadena , Pruebas de Enzimas/métodos , Proteínas Recombinantes/aislamiento & purificación , Reparación del ADN por Recombinación , Ácido Anhídrido Hidrolasas , Animales , Baculoviridae/genética , Tampones (Química) , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/aislamiento & purificación , Enzimas Reparadoras del ADN/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida/instrumentación , Electroforesis en Gel de Poliacrilamida/métodos , Endodesoxirribonucleasas , Pruebas de Enzimas/instrumentación , Humanos , Proteína Homóloga de MRE11/aislamiento & purificación , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Oligonucleótidos/metabolismo , RecQ Helicasas/aislamiento & purificación , RecQ Helicasas/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicación A/aislamiento & purificación , Proteína de Replicación A/metabolismo , Células Sf9 , Spodoptera , Transfección/métodos , Helicasa del Síndrome de Werner/aislamiento & purificación , Helicasa del Síndrome de Werner/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nucleosomas/enzimología , Factores de Transcripción/metabolismo , Animales , Transporte Biológico , ADN/química , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , ADN Recombinante/química , ADN Recombinante/metabolismo , ADN Viral/química , ADN Viral/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Células HeLa , Humanos , Hidrólisis , Cinética , Peso Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Nucleosomas/metabolismo , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
ADN Helicasas/aislamiento & purificación , ADN de Cadena Simple/aislamiento & purificación , Pinzas Ópticas , Imagen Individual de Molécula/métodos , ADN Helicasas/química , ADN de Cadena Simple/química , Escherichia coli/químicaRESUMEN
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.
Asunto(s)
ADN Helicasas/aislamiento & purificación , Microscopía Confocal/métodos , Pinzas Ópticas , Imagen Individual de Molécula/métodos , ADN Helicasas/química , Microscopía Fluorescente/métodos , Nanotecnología/métodosRESUMEN
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.
Asunto(s)
ADN Helicasas/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Nanoporos , Imagen Individual de Molécula/métodos , ADN Helicasas/química , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Nucleótidos , Análisis de Secuencia de ADN/métodosRESUMEN
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.