Saggi biochimici per le attività di analisi ATP-dipendente rimodellamento della cromatina Enzimi
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 complessi di rimodellamento della cromatina famiglia includono un centro SNF2-come ATPasi subunità 1,2. Alcune funzioni ATPasi SNF2-come come enzimi singole subunità, mentre altri funzionano come la subunità catalitica di grandi complessi multi-subunità. Chiarire i meccanismi molecolari attraverso i quali ciascuna delle subunità dei complessi di rimodellamento della cromatina contribuiscono alla loro attività richiede la capacità di effettuare saggi biochimici che sezionano il processo di rimodellamento.
ATP-dipendente rimodellamento dei nucleosomi dal complesso INO80 umano e di altri enzimi rimodellamento della cromatina può essere immaginato come un processo multi-step che inizia con il legame dell'enzima rimodellamento di nucleosomi, seguita dall'attivazione del suo DNA e / o nucleosoma-dipendente ATPasi, traslocazione dell'enzima rimodellamento sul DNA nucleosomal, ed eventuale riposizionamento dei nucleosomi 1,2. Comprendere i dettagli molecolari del ATP-dipendente processo di rimodellamento della cromatina requires dissezione della reazione rimodellamento nelle sue singole fasi e la definizione dei contributi delle singole subunità del complesso rimodellamento della cromatina per ogni fase della reazione. Tali analisi richiedono la capacità di analizzare il rimodellamento dei nucleosomi e altre attività utilizzando substrati molecolari definite in vitro.
In un protocollo JOVE precedente, abbiamo descritto le procedure utilizzate per generare INO80 complessi di rimodellamento della cromatina e Sottocomplessi con composizioni subunità definite 3. Qui, vi presentiamo tre saggi biochimici che permettono l'analisi quantitativa del legame, DNA e nucleosomi attivato ATPasi, e le attività di rimodellamento nucleosoma associati a tali complessi nucleosoma.
Protocol
Representative Results
Discussion
Per garantire che il rimodellamento dei nucleosomi e le attività ATPasi che osserviamo nei test dipendono l'attività catalitica di complessi INO80, e non sulla contaminazione di rimodellamento e / o enzimi ATPasi, abbiamo nucleosomi regolarmente test rimodellamento e l'attività ATPasica di versioni cataliticamente inattive di complessi INO80, purificata in parallelo con wild type INO80 utilizzando la stessa procedura. Una reazione di controllo negativo manca ATP dovrebbe essere eseguita quando saggiando l'…
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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