DNase I Footprint Analysis of Protein‐DNA Binding
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- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
Abstract
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.
Table of Contents
- Basic Protocol 1: DNase I Footprint Titration
- Support Protocol 1: Quantitation of Protein‐Binding Equilibria by Densitometric and Numerical Analyses
- Alternate Protocol 1: DNase Footprinting in Crude Fractions
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol 1: DNase I Footprint Titration
Materials
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Figures
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Figure 12.4.1 Correct positioning of protein‐binding sites from the restriction cuts used to generate a linear, singly end‐labeled fragment. Black boxes represent protein‐binding sites. Asterisks ~undefined) represent [32 P]dNTP incorporated in the Klenow labeling reaction. View Image -
Figure 12.4.2 Footprint titration curve showing the proper range and spacing of protein concentrations. The upper and lower limits of the titration curve are indicated by dashed lines. See Equation for a definition of f . View Image -
Figure 12.4.3 Portion of a footprint titration autoradiogram showing the different blocks that need to be defined for analysis. “Std.” denotes a standard block used to correct for variations in the DNA loaded onto each lane. OI and OE denote protein‐binding sites 1 and 2, respectively. The insert shows regions for a standard block for which background determinations will be made. View Image -
Figure 12.4.4 Individual site‐binding curves determined for a footprint autoradiogram. See Equation for a definition of Y . View Image
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Literature Cited
| Literature Cited | |
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| Ackers, G.K., Shea, M.A., and Smith, F.R. 1983. Free energy coupling within macromolecules: The chemical work of ligand binding at the individual sites in cooperative systems. J. Mol. Biol. 170:223‐242. | |
| Brenowitz, M., Senear, D.F., Shea, M.A., and Ackers, G.K. 1986a. Footprint titrations yield valid thermodynamic isotherms. Proc. Natl. Acad. Sci. U.S.A. 83:8462‐8466. | |
| Brenowitz, M., Senear, D.F., Shea, M.A., and Ackers, G.K. 1986b. Quantitative DNase I footprint titration: A method for studying protein‐DNA interactions. Meth. Enzymol. 130:132‐181. | |
| Dabrowiak, J.C. and Goodisman, J. 1989. Quantitative footprinting analysis of drug‐DNA interactions. In Chemistry and Physics of DNA‐Ligand Interactions (N.R. Kallenback, ed.). Adenine Press. | |
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| Johnson, A.D., Meyer, B.J., and Ptashne, M. 1979. Interactions between DNA‐bound repressors govern regulation by the lambda phage repressor. Proc. Natl. Acad. Sci. U.S.A. 76:5061‐5065. | |
| Johnson, M.L. and Frasier, S.G. 1985. Nonlinear least‐squares analysis. Meth. Enzymol. 117:301‐342. | |
| Senear, D.F., Brenowitz, M., and Ackers, G.K. 1986. Energetics of cooperative protein‐DNA interactions: Comparison between quantitative DNase I footprint titration and filter binding. Biochemistry 25:7344‐7354. | |
| Tullius, T.D., Dombroski, B.A., Churchill, M.E.A., and Kam, L. 1987. Hydroxyl radical footprinting: A high resolution method for mapping protein‐DNA contacts. Meth. Enzymol. 155:537‐558. |
