DNase I Footprint Analysis of Protein‐DNA Binding

Michael Brenowitz1, Donald F. Senear2, Robert E. Kingston3

1 Albert Einstein College of Medicine, Bronx, New York, 2 University of California, Irvine, California, 3 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 12.4
DOI:  10.1002/0471142727.mb1204s07
Online Posting Date:  May, 2001
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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.

     
 
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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
     
 
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Materials

Basic Protocol 1: DNase I Footprint Titration

  Materials
  • Plasmid DNA containing protein‐binding sites (unit 1.6)
  • Appropriate restriction endonucleases (unit 3.1)
  • 100% and ice‐cold 70% ethanol
  • TE buffer ( appendix 22)
  • Aqueous [α‐32P]dNTP (3000 to 6000 Ci/mmol; unit 3.4)
  • 10× Klenow fragment buffer (unit 3.4)
  • Klenow fragment of E. ColiDNA polymerase I (unit 3.5)
  • 5 mM 4dNTP mix (unit 3.4)
  • Deoxyribonuclease I (DNase I; EC 3.1.4.5)
  • recipeAssay buffer A or B
  • Dry ice
  • recipeDNase I stop solution
  • recipeDNase I storage buffer
  • Formamide loading buffer (unit 7.4)
  • 10‐ml plastic disposable tubes
  • Silanized 1.5‐ml microcentrifuge tubes ( appendix 3A)
  • Regulated water bath (±0.1°C)
  • 12 × 7.5‐in. glass or stainless‐steel tray
  • Plastic microcentrifuge tube rack with open sides
  • Gel comb for DNA sequencing gel with 6‐mm lanes spaced on 12‐mm centers
  • Hamilton syringes with blunt‐tip needles (29 G for 0.4‐mm gels)
Additional reagents and equipment for CsCl gradient centrifugation (unit 1.7), restriction enzyme digestion (unit 3.1), ethanol precipitation (unit 2.1), spin column procedure (unit 3.4), agarose gel electrophoresis (unit 2.5), electroelution (unit 2.6), autoradiography ( appendix 3A), denaturing polyacrylamide gel electrophoresis (unit 2.12), and reversed‐phase or ion‐exchange chromatography (Elutip‐d or DEAE‐cellulose, respectively; unit 2.6)
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Figures

Videos

Literature Cited

Literature Cited
   Ackers, G.K., Johnson, A.D., and Shea, M.A. 1982. Quantitative model for gene regulation by lambda phage repressor. Proc. Natl. Acad. Sci. U.S.A. 79:1129‐1133.
   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.
   Galas, D. and Schmitz, A. 1978. DNase footprinting: A simple method for the detection of protein‐DNA binding specificity. Nucl. Acids Res. 5:3157‐3170.
   Hertzberg, R.P. and Dervan, P.B. 1982. Cleavage of double helical DNA by (methidiumpropyl‐EDTA) iron(II). J. Am. Chem. Soc. 104:313‐315.
   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.
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