DNA Footprinting Identifies the Sites Where Proteins Bind DNA

The binding sites for DNA-binding proteins on enhancers and promoters can be identified by a technique called DNA footprinting (Figure 23-12a). In this technique, a cloned DNA fragment containing a putative enhancer or promoter sequence is first radiolabeled at the 5' end with 32P. The labeled DNA is then divided into two fractions: one fraction is incubated with a nuclear extract containing a DNA-binding protein; the other DNA fraction is not incubated with the extract. Both DNA samples are then digested with a nuclease or a chemical that makes random cuts in the phosphodiester bonds of the DNA, and the strands are separated. The resulting DNA fragments are run on a gel to separate fragments of different sizes. In the absence of DNA-binding proteins, a complete ladder of bands is obtained on the electrophoretic gel. When a protein that binds to a site on the DNA fragment is present, it covers some of the nucleotides, protecting that stretch of the DNA from digestion. The electrophoretic pattern of such protected DNA will contain blank regions (or footprints). Each footprint represents the site within an enhancer or promoter that binds a particular DNA-binding protein.

(a) DNA footprinting

(b) Gel-shift analysis

Nuclear extract o o —Sp1

Nuclear extract o o —Sp1

32P r

Sp1 binds to GC box

Protected from cleavage

Footprint

Restriction fragment or oligonucleotide

32pE

Sp1 binds to GC box

Protected from cleavage

DNase I 32P

X-ray film

End-label with 32P

No protein added

Electrophoresis Autoradiography

Restriction fragment or oligonucleotide I I GC | | End-label with 32P

Nuclear extract

DNA without added proteins

Electrophoresis

Autoradiography Specific binding by Sp1

Electrophoresis

Autoradiography Specific binding by Sp1

DNA-protein complex Free DNA fragment

X-ray film

FIGURE 23-12

Identification of DNA sequences that bind protein by DNA-footprinting and gel-shift analysis. (a) In the footprinting technique, labeled DNA fragments containing a putative promoter or enhancer sequence are incubated in the presence and absence of a DNA-binding protein (e.g., Sp1 protein, which binds to a "GC box," a GC-rich region of DNA). After the samples are treated with DNase and the strands separated, the resulting fragments are electrophoresed; the gel then is subjected to autoradiography. A blank region (footprint) in the gel pattern indicates that protein has bound to the DNA. (b) In gel-shift analysis, a labeled DNA fragment is incubated with a cellular extract containing transcription factors. The electrophoretic mobility of the DNA-protein complex is slower than that of free DNA fragments. [Adapted from J. D. Watson et al, 1992, Recombinant DNA, 2nd ed., W. H. Freeman and Company.]

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