Biokemiske analyser til analyse Aktiviteter af ATP-afhængige Kromatin- Remodeling Enzymer
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 kromatin remodeling komplekser indbefatter en central SnF2-lignende ATPase underenhed 1,2. Nogle SnF2-lignende ATPaser funktion som enkelt subunit enzymer, mens andre fungere som den katalytiske underenhed af større multi-subunit-komplekser. Belyse de molekylære mekanismer, som hver af de underenheder af kromatin remodeling komplekser bidrage til deres aktiviteter kræver evnen til at udføre biokemiske analyser, der dissekerer remodeling proces.
ATP-afhængig nukleosom remodellering af den menneskelige INO80 kompleks og andre chromatin remodeling enzymer kan forudses som en multi-trins proces, der begynder med binding af remodeling enzym til nukleosomer, efterfulgt af aktivering af dens DNA- og / eller nukleosom-afhængig ATPase, translokation af ombygningen enzymet på nukleosomal DNA og eventuel repositionering af nucleosomer 1,2. Forståelse af de molekylære detaljer i ATP-afhængig kromatin remodellering proces requires dissektion af ombygningen reaktion i sine enkelte trin og definition af bidrag fra de enkelte underenheder af kromatin remodeling komplekse til hvert trin af reaktionen. Sådanne analyser kræver evnen til at analysere nukleosom ombygninger og andre aktiviteter ved hjælp af definerede molekylære substrater in vitro.
I en tidligere JOVE protokol, vi beskrev procedurer, der anvendes til at generere INO80 kromatin remodeling komplekser og subcomplexes med definerede subunit kompositioner 3. Her præsenterer vi tre biokemiske analyser, der muliggør kvantitativ analyse af nukleosom bindende DNA- og nukleosom aktiveret ATPase, og nukleosom remodeling aktiviteter i forbindelse med sådanne komplekser.
Protocol
Representative Results
Discussion
For at sikre at nukleosom remodeling og ATPase-aktiviteter, vi observerer i assays afhænger af den katalytiske aktivitet af INO80 komplekser, og ikke på kontaminerende remodeling og / eller ATPase-enzymer, vi rutinemæssigt assay nukleosom remodeling og ATPase-aktivitet af katalytisk inaktive versioner af INO80 komplekser oprenses i parallelt med vildtype INO80 efter samme procedure. En negativ kontrol reaktion mangler ATP skal også udføres, når analyse nukleosom remodeling aktivitet at teste for tilstedeværelsen …
Divulgations
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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