10: DNase Footprinting
- Page ID
- 172063
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \)
\( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)
\( \newcommand{\dsum}{\displaystyle\sum\limits} \)
\( \newcommand{\dint}{\displaystyle\int\limits} \)
\( \newcommand{\dlim}{\displaystyle\lim\limits} \)
\( \newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\)
( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\)
\( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\)
\( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\)
\( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\)
\( \newcommand{\Span}{\mathrm{span}}\)
\( \newcommand{\id}{\mathrm{id}}\)
\( \newcommand{\Span}{\mathrm{span}}\)
\( \newcommand{\kernel}{\mathrm{null}\,}\)
\( \newcommand{\range}{\mathrm{range}\,}\)
\( \newcommand{\RealPart}{\mathrm{Re}}\)
\( \newcommand{\ImaginaryPart}{\mathrm{Im}}\)
\( \newcommand{\Argument}{\mathrm{Arg}}\)
\( \newcommand{\norm}[1]{\| #1 \|}\)
\( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\)
\( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\AA}{\unicode[.8,0]{x212B}}\)
\( \newcommand{\vectorA}[1]{\vec{#1}} % arrow\)
\( \newcommand{\vectorAt}[1]{\vec{\text{#1}}} % arrow\)
\( \newcommand{\vectorB}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \)
\( \newcommand{\vectorC}[1]{\textbf{#1}} \)
\( \newcommand{\vectorD}[1]{\overrightarrow{#1}} \)
\( \newcommand{\vectorDt}[1]{\overrightarrow{\text{#1}}} \)
\( \newcommand{\vectE}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash{\mathbf {#1}}}} \)
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \)
\( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)
\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)A DNase footprinting assay is a technique used to determine the specific DNA sequence(s) to which a protein binds in vitro.
Also known as:
DNase I protection assay
DNA footprinting
Samples needed
To perform a DNase footprinting assay, you need:
-
A protein sample, which can be either purified protein or a mixture isolated from cells or tissue.
-
A DNA probe labeled with either a fluorescent or radioactive tag.
-
DNase I or another enzyme that will cleave phosphodiester bonds in the DNA backbone.
Method
In this method, a DNA fragment is radioactively or fluorescently labeled at one end and incubated with the protein of interest. The mixture is then treated with DNase I, an enzyme that randomly cuts the backbone of DNA. The region where the protein is bound is protected from cleavage, creating a "footprint." When the resulting DNA fragments are separated by size using gel electrophoresis, the protected region appears as a gap or missing band in the pattern, revealing the exact binding site of the protein on the DNA. To determine the exact DNA sequence that is protected, Sanger sequencing reactions can be electrophoresed next to the footprinting reactions.
Controls
Typically, a control with DNA + DNase I but no protein is included, because DNase I won’t cleave equally at all DNA sequences. This reaction is compared to DNase I reactions that include protein.
Interpretation
Figure 1. DNase I footprinting assay of the asr promoter. A fluorescently-labeled DNA probe of the asr promoter fragment was incubated with increasing concentrations of purified RstA (lane 1, 0 pmol; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 40 pmol; lane 5, 80 pmol) and subjected to DNase I footprinting assays. Lanes A, T, G, and C represent the respective DNA fragments obtained from Sanger sequencing. The DNA sequence to the right (-77 to -55) is referenced relative to the transcription initiation site. “Figure 3” by Ogasawara et al. [1] [Image description
This figure shows that as the concentration of the RetA protein increases, the DNA sequence from -77 to -55 is protected from cleavage by DNase I. The four leftmost lanes show the results of Sanger sequencing reactions with ddATP, ddGTP, ddTTP, and ddCTP included. The five rightmost lanes show the results of DNase I digestion reactions, with a protected site visible at the two highest concentrations of RetA. The indicated sequence (-77 to -55) is protected from DNase I digestion when bound to the RstA protein, resulting in a lack of bands at the sizes that would correspond to these fragments.
Image Descriptions
Figure 1 image description:
A DNase I footprinting assay. There are nine gel lanes. The first four lanes are Sanger sequencing reactions that can be used to determine the sequence of the asr promoter DNA. The last resulting of Sanger sequencing reactions with ddATP, ddGTP, ddTTP, and ddCTP included. The bands correspond to the DNA sequence of the asr promoter fragment. The last five lanes show the results of DNA fragmentation that is carried out in the presence of DNase I. There is a footprint at the sequence that is bound by the RstA protein (at positions -77 to -55). ↵
Thumbnail
"Binding of SmeT to DNA"↗ by Hernández et al. is licensed under CC BY-SA 4.0↗.
Author
Mitch McVey, Tufts University
1. Ogasawara, H., A. Hasegawa, E. Kanda, T. Miki, K. Yamamoto, and A. Ishihama. 2007. Genomic SELEX search for target promoters under the control of the PhoQP-RstBA single relay cascade. Journal of Bacteriology 189:4791-4799. ↵