ATP에 의존 염색질 리모델링 효소의 활동을 분석하는 생화학 분석 실험
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 가족 염색질 리모델링 단지는 중앙 SNF2 같은 ATPase의 서브 유닛 1, 2를 포함한다. 단일 서브 유니트 효소와 같은 일부 SNF2 형상 ATPases 기능, 다른 큰 멀티 소단위 복합체 촉매 서브 유닛으로서 기능하는 동안. 리모델링 복합체가 그들의 활동에 기여 염색질의 서브 유니트 각각은 리모델링 과정을 해부 생화학 분석을 수행 할 수있는 능력을 필요로하는 분자 메커니즘을 해명.
인간 INO80 착물 및 기타 염색질 개조 효소에 의해 ATP 의존적 뉴 클레오 리모델링은 그 DNA- 및 / 또는 뉴 클레오 의존적 ATPase를 활성화 한 후, 뉴 클레오로 리모델링 효소의 결합을 시작하는 다단계 과정으로 구상 될 수있다 뉴 클레오 솜 DNA, 그리고 뉴 클레오 1,2의 최종 위치 조정에 리모델링 효소의 전위. ATP 의존적 크로 마틴 리모델링 프로세스 (R)의 분자 세부 사항을 이해반응의 각 단계에 크로 마틴 리모델링 복합체의 개별 서브 유닛의 기여의 리모델링의 각 단계에 대한 반응 및 정의의 절개를 equires. 이런 분석은 in vitro에서 정의 된 분자의 기판을 사용하여 뉴 클레오 리모델링 및 기타 활동을 분석 할 수있는 능력을 필요로한다.
이전 조브 프로토콜에서, 우리는 정의 서브 유닛 조성 3 INO80 크로 마틴 리모델링 복합체 및 서브 콤플렉스를 생성하는 데 사용되는 절차를 설명했다. 여기서 우리는, DNA- 및 뉴 클레오-ATPase의 활성화, 이러한 복합체와 관련된 뉴 클레오 리모델링 활동을 결합 뉴 클레오의 정량 분석을 가능하게 세 가지 생화학 적 분석을 제시한다.
Protocol
Representative Results
Discussion
, 그리고 리모델링 및 / 또는 ATPase의 효소 오염에 우리가 분석에서 관찰하는 뉴 클레오 리모델링 및 ATPase의 활동 INO80 복합체의 촉매 활성에 따라 보장하기 위해, 우리는 정기적으로 분석 뉴 클레오 리모델링 및 INO80 단지의 촉매 비활성 버전의 ATPase의 활동은 정제 야생형 INO80 동일한 절차를 사용하여 평행. INO80 착체 또는 서브 콤플렉스의 다른 제제의 액티비티를 비교하는 실험의 해석을 복잡하게…
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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