Biochemischen Assays zur Analyse von Aktivitäten von ATP-abhängigen Chromatin-Remodeling-Enzyme
Summary
Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.
Abstract
Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.
Introduction
SNF2 Familie Chromatin-Remodeling Komplexe umfassen eine zentrale SNF2-ATPase-Untereinheit wie 1,2. Einige SNF2 artigen ATPasen Funktion als einzelne Untereinheit Enzyme, andere Funktion als die katalytische Untereinheit von größeren Multi-Untereinheiten-Komplexen. Aufklärung der molekularen Mechanismen, durch die jede der Untereinheiten von Chromatin-Remodeling-Komplexe beitragen, ihre Tätigkeit erfordert die Fähigkeit, biochemische Assays, die die Umbauprozess auszuführen sezieren.
ATP-abhängige Nukleosom-Remodeling vom menschlichen INO80 Komplex und andere Chromatin-Remodeling-Enzyme können nach einem mehrstufigen Prozess, der mit der Bindung des Enzyms an Nukleosomen Umbau beginnt in Betracht gezogen werden, gefolgt von der Aktivierung der DNA-und / oder Nukleosom-abhängige ATPase, Translokation des Enzyms auf Umbau nukleosomalen DNA und eventuelle Neupositionierung der Nukleosomen 1,2. Das Verständnis der molekularen Details der ATP-abhängigen Chromatin-Remodeling-Prozess requires Dissektion der Umbau Reaktion in seine einzelnen Schritte des Wortes der Beiträge der einzelnen Untereinheiten des Chromatin-Remodeling-Komplexes, um jeden Schritt der Reaktion. Solche Analysen erfordern die Fähigkeit zur Analyse Nukleosomen Umbau-und andere Aktivitäten mit definierten molekularen Substrate in vitro.
In einem früheren JOVE Protokoll beschrieben wir Verfahren verwendet werden, um INO80 Chromatin-Remodeling-Komplexe und Subkomplexe mit definierten Untereinheit 3 Kompositionen zu erzeugen. Hier präsentieren wir drei biochemischen Assays, die quantitative Analyse der Nukleosomen-Bindung, DNA und Nukleosomen-ATPase aktiviert, und Nukleosomen Umbau Aktivitäten mit solchen Komplexen assoziiert zu ermöglichen.
Protocol
Representative Results
Discussion
Um sicherzustellen, dass Nukleosomen Umbau-und ATPase-Aktivitäten, die wir in Tests zu beobachten, hängt von der katalytischen Aktivität des INO80 Komplexe und nicht auf kontaminierende Umbau-und / oder ATPase-Enzyme, die wir routinemßig Assay Nukleosom Umbau-und ATPase-Aktivität der katalytisch inaktiven Versionen INO80 Komplexe, gereinigt parallel mit Wildtyp-INO80 mit dem gleichen Verfahren. Eine negative Kontrollreaktion fehlt ATP sollte auch bei der Untersuchung von Nukleosomen Umbau Aktivität, um das Vorhand…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Work in the authors’ laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.
Materials
| Name of Reagent/Material | Company | Catalog Number | Comments |
| Protease Inhibitor Cocktail | Sigma | P8340 | |
| 10x PCR reaction buffer | Roche Applied Science | 11435094001 | |
| Roche Taq DNA Polymerase | Roche Applied Science | 11435094001 | |
| NucAway Nuclease-free Spin Columns | Ambion | Cat. # AM10070 | |
| ultrapure ATP | USB/Affymetrix | 77241 25 UM | |
| bovine serum albumin | Sigma | A9418 | |
| N,N,N´,N´-tetramethylethylenediamine (TEMED) | Thermo Scientific | 17919 | Fisher Scientific |
| 40% Acrylamide/Bis 37.5:1 | Amresco | 0254-500ML | |
| Sonicated salmon sperm DNAs | GE Healthcare | 27-4565-01 | |
| 10% ammonium persulfate (APS) | Thermo Scientific | 17874 | |
| benzonase | Novagen | Cat. No. 70664 | |
| [α-32P] ATP (3000 Ci/mmol) | PerkinElmer | BLU003H250UC | |
| dCTP, [α-32P]- 6000Ci/mmol | PerkinElmer | BLU013Z250UC | |
| Equipment | Company | ||
| PCR thermal cycler PTC 200 | MJ Research | PTC 200 | |
| Hoefer vertical electrophoresis unit | Hoefer | SE600X-15-1.5 | |
| lubricated 1.5ml microcentrifuge tubes | Costar | 3207 | |
| Storage Phosphor Screen | Molecular Dynamics | 63-0034-79 | |
| 3MM filter paper | Whatman | 28458-005 | VWR |
| Typhoon PhosphorImager | GE Healthcare | 8600 | |
| ImageQuant software | GE Healthcare | ver2003.02 | |
| TLC Glass Plates, PEI-Cellulose F | Millipore | 5725-7 | |
| Immobilon-FL Transfer Membrane 7 x 8.4 | Millipore | IPFL07810 | |
| General purpose survey meter with end-window or pancake GM (Geiger-Mueller) probe | Biodex | Model 14C |
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