Unit

UNIT 12.4 DNase I Footprint Analysis of Protein-DNA Binding

  1. Michael Brenowitz1,
  2. Donald F. Senear2,
  3. Robert E. Kingston3

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb1204s07

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Brenowitz, M., Senear, D. F. and Kingston, R. E. 2001. DNase I Footprint Analysis of Protein-DNA Binding. Current Protocols in Molecular Biology. 7:12.4:12.4.1–12.4.16.

Author Information

  1. 1

    Albert Einstein College of Medicine, Bronx, New York

  2. 2

    University of California, Irvine, California

  3. 3

    Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JUL 1989

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 Support Protocol 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 Alternate Protocol describes the qualitative use of footprinting to identify DNA-binding proteins in crude extracts.