एटीपी निर्भर Chromatin Remodeling एंजाइमों की गतिविधियों का विश्लेषण के लिए जैव रासायनिक assays
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 परिवार chromatin remodeling परिसरों एक केंद्रीय SNF2 तरह ATPase सबयूनिट 1,2 शामिल हैं. एकल सबयूनिट एंजाइमों के रूप में कुछ SNF2 तरह ATPases समारोह, दूसरों बड़ा बहु सबयूनिट परिसरों का उत्प्रेरक सबयूनिट के रूप में कार्य करते हुए. Remodeling परिसरों उनकी गतिविधियों में योगदान क्रोमेटिन के सब यूनिटों में से प्रत्येक remodeling प्रक्रिया काटना कि जैव रासायनिक assays प्रदर्शन करने की क्षमता की आवश्यकता है जिसके द्वारा आणविक तंत्र elucidating.
मानव INO80 परिसर और अन्य chromatin remodeling एंजाइमों द्वारा एटीपी निर्भर nucleosome remodeling, इसके DNA- और / या nucleosome निर्भर ATPase के सक्रियण द्वारा पीछा किया, nucleosomes को remodeling एंजाइम के बंधन के साथ शुरू होता है कि एक बहु कदम प्रक्रिया के रूप में कल्पना की जा सकती है nucleosomal डीएनए, और nucleosomes 1,2 के अंतिम repositioning पर remodeling एंजाइम का स्थानान्तरण. एटीपी निर्भर chromatin remodeling प्रक्रिया आर के आणविक विवरण को समझनाप्रतिक्रिया के हर कदम को chromatin remodeling जटिल की व्यक्तिगत सब यूनिटों के योगदान के पुनर्गठन अपनी अलग अलग चरणों में प्रतिक्रिया और परिभाषा के विच्छेदन equires. इस तरह के विश्लेषण के लिए इन विट्रो में परिभाषित आणविक substrates का उपयोग nucleosome remodeling और अन्य गतिविधियों का विश्लेषण करने की क्षमता की आवश्यकता होती है.
पिछले एक जौव प्रोटोकॉल में, हम परिभाषित सबयूनिट रचनाओं 3 के साथ INO80 chromatin remodeling परिसरों और subcomplexes उत्पन्न करने के लिए प्रयोग किया जाता प्रक्रियाओं का वर्णन किया. यहाँ, हम, DNA- और nucleosome सक्रिय ATPase, और इस तरह के परिसरों के साथ जुड़े nucleosome remodeling गतिविधियों बंधन nucleosome की मात्रात्मक विश्लेषण कर सकें कि तीन जैव रासायनिक assays प्रस्तुत करते हैं.
Protocol
Representative Results
Discussion
, और नहीं remodeling और / या ATPase एंजाइम contaminating पर हम assays में निरीक्षण कि nucleosome remodeling और ATPase गतिविधियों INO80 परिसरों का उत्प्रेरक गतिविधि पर निर्भर सुनिश्चित करने के लिए, हम नियमित तौर पर परख nucleosome remodeling और INO80 परिसरों की catalytically न?…
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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