Los ensayos bioquímicos para Actividades Analizando de ATP-dependientes remodelación de la cromatina enzimas
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
Complejos remodeladores de cromatina familia SNF2 incluyen una SNF2 como ATPasa subunidad central de 1,2. Algunos ATPasas función como SNF2 como enzimas de subunidades individuales, mientras que otros funcionan como la subunidad catalítica de complejos de múltiples subunidades más grandes. Dilucidar los mecanismos moleculares por los cuales cada una de las subunidades de la cromatina complejos remodeladores de contribuir a sus actividades requiere la capacidad de realizar ensayos bioquímicos que diseccionan el proceso de remodelación.
Remodelación nucleosoma dependiente de ATP por el complejo INO80 humana y otras enzimas remodelación de la cromatina puede ser concebido como un proceso de múltiples pasos que comienza con la unión de la enzima a la remodelación nucleosomas, seguido por la activación de su ADN y / o nucleosoma-ATPasa dependiente, translocación de la enzima sobre el ADN nucleosomal remodelación, y la eventual reposicionamiento de los nucleosomas 1,2. La comprensión de los detalles moleculares de la ATP-dependiente proceso de remodelación de la cromatina requiere disección de la reacción de remodelación en sus pasos individuales y definición de las contribuciones de las subunidades individuales del complejo de remodelación de la cromatina a cada etapa de la reacción. Tales análisis requieren la capacidad de analizar la remodelación nucleosoma y otras actividades utilizando sustratos moleculares definidos in vitro.
En un protocolo JOVE anterior, describimos los procedimientos utilizados para generar complejos remodeladores de cromatina INO80 y subcomplexes con composiciones de subunidades definidas 3. A continuación, presentamos tres ensayos bioquímicos que permiten el análisis cuantitativo de la unión, ADN y-nucleosoma activado ATPasa, y actividades de remodelación de nucleosomas asociados con tales complejos de nucleosomas.
Protocol
Representative Results
Discussion
Para asegurarse de que la remodelación nucleosoma y actividades de ATPasa se observa en los ensayos dependen de la actividad catalítica de complejos INO80, y no en la contaminación de la remodelación y / o enzimas ATPasa, que nucleosoma ensayo de forma rutinaria la remodelación y la actividad ATPasa de versiones catalíticamente inactivos de Ino80, purificado en en paralelo con el tipo salvaje INO80 utilizando el mismo procedimiento. Una reacción de control negativo que carece de ATP también se debe realizar cuan…
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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