ATP-bağımlı Kromatin Tadilat Enzimler Faaliyetlerinin Analizi için biyokimyasal Tahliller
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 aile kromatin biçimlenme kompleksleri merkezi SnF2 gibi ATPaz alt birimini 1,2 içerir. Tek alt-birim enzimler gibi bazı SnF2 gibi ATPaz fonksiyonu, diğer daha büyük bir çoklu alt birim kompleksi katalitik alt-birimi olarak işlev yaparken. Biçimlenme kompleksleri onların faaliyetlerine katkıda kromatinin alt birimlerinin her biçimlenme sürecini incelemek biyokimyasal analizleri gerçekleştirmek için yeteneği gerektiren hangi moleküler mekanizmaların tanıtılması.
İnsan INO80 kompleksi ve diğer enzimler tarafından yeniden yapılanma kromatin ATP'ye bağımlı nükleozom yeniden modelleme, kendi DNA- ve / veya nükleozom bağımlı ATPaz aktivasyonu takiben, nükleozomlann için yeniden şekillenme enzimin bağlanması ile başlayan bir çok-aşamalı bir süreç olarak tasavvur edilebilir nükleozomal DNA ve nükleozomlardan 1,2 nihai konumlandırılması hakkında yeniden şekillenme enzim translokasyon. ATP'ye bağımlı kromatin değiştirme prosesinin r molekül ayrıntılarını anlamakreaksiyonun her aşaması için kromatin remodeling karmaşık bireysel alt birimlerinin katkıları biçimlenme kendi bireysel adımlar içine reaksiyonu ve tanımı diseksiyonu equires. Bu tür analizler in vitro tanımlanmış moleküler alt tabakalar kullanılarak nucleosome biçimlenme ve diğer faaliyetlerini analiz yeteneği gerektirir.
Önceki bir JOVE protokol, tanımlanmış ait birim bileşimleri ile 3 INO80 kromatin biçimlenme kompleksleri ve subcomplexes oluşturmak için kullanılan prosedürleri tarif. Burada, biz, DNA'ya ve nucleosome-aktive ATPaz ve bu kompleksleri ile ilişkili nucleosome biçimlenme faaliyetlerini bağlayıcı nucleosome kantitatif analizini sağlayan üç biyokimyasal analizler sunmak.
Protocol
Representative Results
Discussion
, Ve yeniden modelleme ve / veya enzimler ATPaz kirletici biz deneylerde uyulması, nükleozom şekillenmesi ve ATPaz aktiviteleri INO80 komplekslerin katalitik aktivitesine bağlı sağlamak için, rutin deney nükleozom modelleme ve INO80 komplekslerin katalitik olarak aktif olmayan ATPaz aktivitesi, saflaştırılır Yabani tip INO80 aynı prosedür kullanılarak paralel. INO80 kompleksi ya da subcomplexes farklı preparatlar içinde etkinliklerini karşılaştırmak, deney yöntemi için karmaşık hale ATP ve / vey…
Declarações
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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