El desarrollo del ADN se extiende ensayo fue ayudado por Paul Blainey, Bong Jong-Lee, y Samir Hamdan. Este trabajo es apoyado por los Institutos Nacionales de Salud subvenciones a Charles Richardson (GM54397), y Antoine van Oijen (GM077248).

<ol>
	<li>Lee, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+primase+acts+as+a+molecular+brake+in+DNA+replication.">DNA primase acts as a molecular brake in DNA replication.</a> Nature. 439, 621-624 (2006).</li><li>Tanner, N. A., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+observation+of+prokaryotic+DNA+replication.">Single-molecule observation of prokaryotic DNA replication.</a> Methods Mol Biol. 521, 397-410 (2009).</li><li>Sofia, S. J., Premnath, V. V., Merrill, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ethylene+oxide)+Grafted+to+Silicon+Surfaces:+Grafting+Density+and+Protein+Adsorption.">Poly(ethylene oxide) Grafted to Silicon Surfaces: Grafting Density and Protein Adsorption.</a> Macromolecules. 31, 5059-5070 (1998).</li><li>Tanner, N. A., Loparo, J. J., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+single-molecule+DNA+replication+with+fluorescence+microscopy.">Visualizing single-molecule DNA replication with fluorescence microscopy.</a> J Vis Exp. , (2009).</li><li>Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+DNA+replication+loops+reveal+temporal+control+of+lagging-strand+synthesis.">Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.</a> Nature. 457, 336-339 (2009).</li><li>van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+kinetics+of+lambda+exonuclease+reveal+base+dependence+and+dynamic+disorder.">Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder.</a> Science. 301, 1235-1238 (2003).</li></ol>

Es importante asegurarse de que la fuerza de arrastre ejercida sobre cuentas de flujo laminar no influye en la actividad enzimática de las proteínas de replicación. Por ejemplo, una fuerza de 3 PN que corresponde a un caudal de 0.012 ml / min no afectan a la replicación de la hebra de ADN líder. Sin embargo, sí afecta a las actividades enzimáticas que se producen durante la síntesis de ADN coordinado, y por lo tanto, tiene que reducirse a 1,5 pN. La fuerza de arrastre puede ser fácilmente controlado por el camb...

<table><tbody><tr><td>Bacteriophage &amp;#955; DNA</td><td>New England Biolabs</td><td>N3011L</td><td></td></tr><tr><td>DNA Oligonucleotides</td><td>Integrated DNA Technologies</td><td></td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202L</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>New England Biolabs</td><td>M0201L</td><td></td></tr><tr><td>&amp;alpha;-digoxigenin Fab</td><td>Roche Group</td><td>11214667001</td><td></td></tr><tr><td>Tosyl Activated Beads</td><td>Invitrogen</td><td>142-03</td><td></td></tr><tr><td>Magnetic Separator</td><td>Invitrogen</td><td>Dynal MPC</td><td></td></tr><tr><td>3-aminopropyl-triethoxysilane</td><td>Sigma-Aldrich</td><td>A3648</td><td>Other aminosilanes can be used or mixed with non-amine reactive silanes for sparser surfaces</td></tr><tr><td>Succinimidyl propionate PEG</td><td>Nektar</td><td></td><td>Similar PEGs can be purchased from Nanocs, CreativePEGWorks, etc.</td></tr><tr><td>Biotin-PEG-NHS</td><td>Nektar</td><td></td><td>Similar PEGs can be purchased from Nanocs, CreativePEGWorks, etc.</td></tr><tr><td>Double-sided tape</td><td>Grace Bio-Lab Inc.</td><td>SA-S-1L</td><td>100 &amp;#x03BC;m thickness</td></tr><tr><td>Quartz slide</td><td>Technical Glass</td><td>20 mm (W)x 50 mm (L)x 1mm (H)</td><td>Size to fit on coverslips. Drill holes with diamond-tip drill bits (DiamondBurs.net)</td></tr><tr><td>Polyethylene tubing</td><td>BD Biosciences</td><td>427416</td><td>0.76 mm ID, 1.22 OD&lt;br/&gt;Other size tubing can be substituted.</td></tr><tr><td>Streptavidin</td><td>Sigma-Aldrich</td><td>S4762</td><td>Make 1 mg/mL solution, 25 &amp;#x03BC;L aliquots in PBS pH 7.3</td></tr><tr><td>Deoxyribonucleotide triphosphate solution mix</td><td>New England Biolabs</td><td>N0447</td><td></td></tr><tr><td>Inverted Optical Microscope with 10X Objective</td><td>Olympus Corporation</td><td>Olympus IX-51</td><td></td></tr><tr><td>Permament rare-earth magnet</td><td>National Imports</td><td>www.rare-earth-magnets.com</td><td></td></tr><tr><td>CCD Camera</td><td>QImaging</td><td>Rolera-XR Fast 139</td><td></td></tr><tr><td>Syringe Pump</td><td>Harvard Apparatus</td><td>11 Plus</td><td>Operate in refill mode to facilitate solution changes</td></tr><tr><td>Fiber Illuminator</td><td>Thorlabs Inc.</td><td>OSL1</td><td></td></tr></tbody></table>

direct observation of enzymes replicating dna using a single-molecule dna stretching assay

Se describe un método para observar la replicación en tiempo real de las moléculas individuales de ADN mediada por proteínas del sistema de replicación del bacteriófago. Linealizado λ ADN se modifica para tener un biotina en el extremo de un hilo, y una fracción de digoxigenina en el otro extremo de la misma cadena. El final con biotina se une a un cubreobjetos funcionalizados y al final digoxigeninated a una pequeña cuenta. El montaje de estas correas de ADN derramará en la superficie de una célula de flujo permite un flujo laminar que se aplicarán para ejercer una fuerza de arrastre sobre el talón. Como resultado, el ADN se estira cerca y en paralelo a la superficie del cubreobjetos en una fuerza que está determinada por la velocidad de flujo (Figura 1). La longitud del ADN se mide mediante el control de la posición de la bola. Las diferencias de longitud entre el ADN de una sola y de doble cadena se utilizan para obtener información en tiempo real sobre la actividad de las proteínas de la replicación en el tenedor. Medición de la posición de la bola permite la determinación precisa de las tasas y processivities de anulación de ADN y la polimerización (Figura 2).

Watch this Scientific Journal Video about Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay at JoVE.com

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

Se describe un método para observar la replicación en tiempo real de las moléculas individuales de ADN mediada por proteínas del sistema de replicación del bacteriófago.

La observación directa de ADN Enzimas Replicar El uso de un ADN de una sola molécula de estiramiento de ensayo

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

direct-observation-enzymes-replicating-dna-using-single-molecule-dna

Research

JoVE Journal

Biology

18.7K vistas.  Harvard Medical School. Se describe un método para observar la replicación en tiempo real de las moléculas individuales de ADN mediada por proteínas del sistema de replicación del bacteriófago.

Video: Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

Una sola molécula Simple, robusto y alto rendimiento flujo estiramiento aplicación de análisis para el estudio de transporte de moléculas a lo largo de la DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Investigación

Bioquímica

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

Ensayo de actividad polimerasa de la DNA usando la DNA etiquetado fluorescente infrarroja visualizado por electroforesis en Gel de acrilamida

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

Revisión y análisis de la reparación de ADN usando análisis de extensión de un solo nucleótido y MALDI-TOF espectrometría de masa

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

Genética

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualización de una sola molécula de ADN de replicación con microscopía de fluorescencia

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Biología

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

Análisis de tiempo de permanencia de una sola molécula de la escisión de ADN mediada por endonucleasas de restricción

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Obtención de imágenes del movimiento de Cdc45/Mcm2-7/GINS (CMG) completamente reconstituido

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Autor destacado: Investigación de la dinámica de movimiento de los componentes replisómicos eucariotas a nivel de una sola molécula

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

Ensayo híbrido de conjunto y molécula única para obtener imágenes del movimiento de la CMG totalmente reconstituida

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication ...

<meta charset="utf8"></head><body>Desenrollamiento del ADN mediado por CMG observado por el crecimiento del tracto ssDNA-RPA

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

<meta charset="utf8"></head><body>Autor destacado: Desentrañando la dinámica de la replicación del ADN eucariota a través de la visualización de una sola molécula

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Visualización en tiempo real de una sola molécula del desenrollado del ADN mediante CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

La observación directa de ADN Enzimas Replicar El uso de un ADN de una sola molécula de estiramiento de ensayo

Resumen

Explorar más videos

Capítulos en este video

Una sola molécula Simple, robusto y alto rendimiento flujo estiramiento aplicación de análisis para el estudio de transporte de moléculas a lo largo de la DNA

Ensayo de actividad polimerasa de la DNA usando la DNA etiquetado fluorescente infrarroja visualizado por electroforesis en Gel de acrilamida

Revisión y análisis de la reparación de ADN usando análisis de extensión de un solo nucleótido y MALDI-TOF espectrometría de masa

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Visualización de una sola molécula de ADN de replicación con microscopía de fluorescencia

Análisis de tiempo de permanencia de una sola molécula de la escisión de ADN mediada por endonucleasas de restricción

Ensayo híbrido de conjunto y molécula única para obtener imágenes del movimiento de la CMG totalmente reconstituida

Ensayo híbrido de conjunto y molécula única para obtener imágenes del movimiento de la CMG totalmente reconstituida

Ensayo híbrido de conjunto y molécula única para obtener imágenes del movimiento de la CMG totalmente reconstituida

Visualización en tiempo real de una sola molécula del desenrollado del ADN mediante CMG Helicase