Biokemiska analyser för att analysera aktiviteter av ATP-beroende Chromatin 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 familjen kromatin remodeling komplex innefattar en central SnF2 liknande ATPas subenhet 1,2. Vissa SnF2 liknande ATPaser funktion som enskilda subenheten enzymer, medan andra fungerar som den katalytiska subenheten av större multi subenhet komplex. Belysa de molekylära mekanismer genom vilka var och en av subenheterna av kromatin remodeling komplex bidrar till deras verksamhet kräver förmågan att utföra biokemiska analyser som dissekera remodelleringsprocessen.
ATP-beroende nukleosomen remodellering av den mänskliga INO80 komplex och andra kromatin remodeling enzymer kan förutses som en flerstegsprocess som börjar med bindning av remodeling enzym till nukleosomer, följt av aktivering av dess DNA- och / eller nukleosom-beroende ATPas, translokation av ombyggnad enzym på nucleosomal DNA, och eventuell omplacering av nukleosomer 1,2. Förstå de molekylära detaljerna i ATP-beroende kromatin remodelleringsprocessen requires dissektion av ombyggnad reaktion till dess enskilda åtgärder och fastställande av bidragen från enskilda subenheter av kromatin remodeling komplex för varje steg i reaktionen. Sådana analyser kräver förmåga att analysera nukleosomen ombyggnad och andra aktiviteter med hjälp av definierade molekylära substrat in vitro.
I en tidigare JOVE protokoll, beskrev vi metoder som används för att generera INO80 kromatin remodeling komplex och subcomplexes med definierade subenheten kompositioner 3. Här presenterar vi tre biokemiska analyser som möjliggör kvantitativ analys av nukleosomen bindande, DNA- och nukleosom-aktiverade ATPas och nukleosomen remodeling verksamhet i samband med sådana komplex.
Protocol
Representative Results
Discussion
För att säkerställa att nukleosomen ombyggnad och ATPas aktiviteter vi observerar i analyser är beroende av den katalytiska aktiviteten av INO80 komplex, och inte på kontamine remodeling och / eller ATPas enzymer, vi rutinmässigt analys nukleosomen ombyggnad och ATPas aktivitet katalytiskt inaktiva versioner av INO80 komplex, renas i parallellt med vildtyp INO80 enligt samma förfarande. En negativ kontrollreaktion som saknar ATP bör också göras vid analys nukleosomen remodeling aktivitet för att undersöka f?…
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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