ATP依存性クロマチンリモデリング酵素の活性を分析するための生化学的アッセイ
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家族のクロマチンリモデリング複合体は、中央SNF2様ATPアーゼサブユニット1,2を含む。単一のサブユニットの酵素など、一部SNF2様ATPアーゼ機能、他の人がより大きなマルチサブユニット複合体の触媒サブユニットとして機能している。クロマチンリモデリング複合体のサブユニットのそれぞれが自分の活動に貢献する分子メカニズムを解明することリモデリングプロセスを分析生化学的アッセイを行う能力が必要です。
人間INO80複合体および他のクロマチンリモデリング酵素によるATP依存ヌクレオソームのリモデリングは、その、DNAおよび/またはヌクレオソーム依存ATPaseの活性化に続いて、ヌクレオソームのリモデリング酵素の結合で始まる多段階のプロセスとして考えることができるヌクレオソームDNA上のリモデリング酵素の転座、およびヌクレオソーム1,2の最終的な再配置。 ATP依存性クロマチンリモデリングプロセスrの分子の詳細を理解するその個別のステップや反応の各ステップにクロマチンリモデリング複合体の各サブユニットの寄与の定義にリモデリング反応の解剖をequires。このような分析は、 インビトロで規定された分子基質を用いてヌクレオソームのリモデリングおよび他の活動を分析する能力を必要とする。
以前JOVEプロトコルでは、定義されたサブユニット組成物3とINO80クロマチンリモデリング複合体とsubcomplexesを生成するために使用される手順を説明した。ここでは、3ヌクレオソーム結合、DNAおよびヌクレオソーム活性化ATPアーゼの定量分析を可能にする生化学的アッセイ、およびこのような複合体に関連したヌクレオソームのリモデリング活動を紹介します。
Protocol
Representative Results
Discussion
確実にするために私たちはアッセイで観察することがヌクレオソームのリモデリングおよびATPアーゼ活性はINO80複合体の触媒活性にではなく、中で精製INO80複合体の触媒的に不活性なバージョンの改造を汚染し、および/またはATPアーゼの酵素、私たちは日常的にアッセイヌクレオソームのリモデリングとATPase活性に依存同じ手順を使用して、野生型INO80と平行である。 INO80複合体またはsubcomple…
Offenlegungen
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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