DNase I footprint analysis of protein-DNA binding.
DNase I footprint analysis of protein-DNA binding.
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DOI：
10.1002/0471142727.mb1204s07
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发表时间：
2001-05-01
影响因子：
--
通讯作者：
Kingston, R E
中科院分区：
文献类型：
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作者：
Brenowitz, M;Senear, D F;Kingston, R E
脱氧核糖核酸酶I （DNase I）保护定位或足迹定位是一种有价值的技术，用于定位蛋白质在DNA上的特定结合位点。这个实验的基础是结合蛋白保护DNA的磷酸二酯主链免受DNA酶i催化水解。结合位点是通过对水解产生的DNA片段进行放射自显影，然后在变性DNA测序凝胶上进行电泳分离。足迹作为一种定量技术已经得到了进一步的发展，以确定DNA上每个单独的蛋白质结合位点的单独结合曲线。对于每个结合位点，结合的总能量直接由该位点的结合曲线确定。对于协同相互作用的位点，同时对所有结合曲线进行数值分析，可以求解出这些能量的本征结合和协同分量。本单元描述了DNase I足迹滴定，包括(1)制备单端标记的DNA限制片段，(2)蛋白质与DNA的平衡，(3)将平衡混合物暴露于DNase I，以及(4)变性水解产物凝胶上的电泳分离，然后进行放射自显影。A描述了(1)对自射线图进行密度分析，以获得结合数据；(2)对结合数据进行数值分析，得出每个单独位点上相互作用的结合曲线和平衡常数。An描述了在粗提取物中定性地使用足迹来鉴定dna结合蛋白。
Deoxyribonuclease I (DNase I) protection mapping, or footprinting, is a valuable technique for locating the specific binding sites of proteins on DNA. The basis of this assay is that bound protein protects the phosphodiester backbone of DNA from DNase I-catalyzed hydrolysis. Binding sites are visualized by autoradiography of the DNA fragments that result from hydrolysis, following separation by electrophoresis on denaturing DNA sequencing gels. Footprinting has been developed further as a quantitative technique to determine separate binding curves for each individual protein-binding site on the DNA. For each binding site, the total energy of binding is determined directly from that site's binding curve. For sites that interact cooperatively, simultaneous numerical analysis of all the binding curves can be used to resolve both the intrinsic binding and cooperative components of these energies.DNase I footprint titration is described in this unit and involves (1) preparation of a singly end-labeled DNA restriction fragment, (2) equilibration of the protein with DNA, (3) exposure of the equilibrium mixture to DNase I, and (4) electrophoretic separation on gels of the denatured hydrolysis products, followed by autoradiography. A describes (1) densitometric analysis of the autoradiograms to obtain binding data and (2) numerical analysis of the binding data to yield binding curves and equilibrium constants for the interactions at each of the separate sites. An describes the qualitative use of footprinting to identify DNA-binding proteins in crude extracts.