Moss Tom. DNA-protein interactions: principles and protocols
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Use of DEPC and Potassium Permanganate as Probes 67
sections are general guidelines for probing with DEPC and potassium perman-
ganate. Care and attention to detail is necessary for all of these methods, as
footprinting with these reagents is generally somewhat more difficult than with
many other footprinting reagents. There is usually a need to optimize condi-
tions for each particular application; guidelines for this can be found in the
Notes section.
2. Materials
1. Potassium permanganate, DEPC, piperidine, and 2-mercaptoethanol can be pur-
chased from Sigma (St. Louis, MO). DEPC, piperidine, and 2-mercaptoethanol
should be stored at 4°C and used with caution in a fume hood. Other reagents
listed below should be of the highest quality. Low-adhesion microcentrifuge tubes
(siliconized) can be obtained from USA Scientific (Ocala, FL).
2. The following stock solutions can be made, filter sterilized, divided into aliquots
and stored at –20°C until ready for use:
1 M HEPES pH 7.9
1 M MgCl
2
0.5 M dithiothreitol (DTT)
2 M KCl
10 mg/mL bovine serum albumin (BSA), DNase free
0.5 M EDTA pH 8.0
3 M Sodium acetate pH 5.2
5 mg/mL linear polyacrylamide (acrylamide polymerized without N,N'-methylene
bisacrylamide)
10 mg/mL Proteinase K
10% sodium dodecyl sulfate (SDS)
DEPC stop buffer: 0.2% SDS and 0.6 M sodium acetate pH 5.2
TE: 10 mM Tris–HCl pH 8.0 and 1 mM EDTA
Electrophoresis loading buffer: 0.1% bromophenol blue, 0.1% xylene cyanol,
10 mM EDTA, and 80% deionized formamide
3. A 300 mM stock solution of KMnO
4
can be made by heating 2.73 g of KMnO
4
in
50 mL of deionized water. The solution can be stored at room temperature in a
brown bottle for 1 mo.
4. 10X reaction buffer, for example: 200 mM HEPES pH 7.9, 100 mM MgCl
2
, 1 mM
DTT, 1 mg/mL BSA, and 100 mM KCl. The choice of this buffer will depend on
the conditions needed to form the complex under study.
5. Protein dilution buffer, for example: 20 mM HEPES pH 7.9, 1 mM EDTA, and
10% glycerol. Choice will depend on protein used.
6. 1 M piperidine: 10% v/v in water, freshly made.
7. 0.6 M sodium acetate.
8. 0.6 M sodium acetate, 20 mM EDTA
9. 10X neutralization buffer: 0.5 M HEPES pH 7.9, 0.1 M MgSO
4
, and 2 mM DTT.
10. dNTP mix: 5 mM of each dNTP.
68 Kahl and Paule
3. Methods
Experiments using DEPC or potassium permanganate follow similar proto-
cols. The only exceptions are the addition of 2-mercaptoethanol to quench
reactions using potassium permanganate, the duration of the DNA modifica-
tion reactions, and the necessity to purify the modified DNA away from DEPC
before the cleavage step.
3.1. In Vitro Experiments on Linear DNA Fragments
1. End-labeled DNA (5' or 3') should be separated from excess radiolabeled precur-
sor either by agarose gel electrophoresis or size-exclusion column chromatogra-
phy, and stored in TE.
2. In a 1.5-mL siliconized microcentrifuge tube, add 4 µL 10X reaction buffer,
40,000 cpm of DNA, and sterile deionized water to a volume of 20 µL. Proteins,
diluted in an appropriate buffer, are added to the reaction to give a final volume
of 40 µL (see Note 1). Incubate for the desired amount of time to form DNA–
protein complexes.
3. Modifying the susceptible bases (see Notes 2–4):
KMnO
4
treatment: Add freshly diluted potassium permanganate to give the
appropriate concentration and incubate for desired time period. For
example, adding 2 µL of 100 mM KMnO
4
(approximately 9 mM final con-
centration) for 2 min seems to work well for detecting melted DNA in
transcription initiation complexes.
Diethyl procarbonate treatment: Add 1 µL of DEPC to each tube. Mix by
briefly vortexing and repeat vortexing every 5 min for 15 min. Vortexing
is necessary because DEPC is sparingly soluble in aqueous solutions, so
all reactions are run at essentially saturating DEPC and the concentration
cannot be altered significantly.
4. Stopping the reactions:
KMnO
4
treatment: Quench the reaction by adding 3 µL of 2-mercaptoethanol,
vortex and place on ice. Add 45 µL of 0.2% SDS and 2 mg/mL proteinase
K and incubate at 50°C for 1 h. Add 90 µL of 0.6 M sodium acetate,
300 µg/mL linear polyacrylamide, and 2.5 volumes 95% ethanol. Mix and
centrifuge 30 min at 14,000 rpm in a microfuge. Remove supernatant and
wash with 150 µL of 70% ethanol. Centrifuge 5 min, as above. Remove
supernatant and dry pellet on medium heat for 5 min in a Speed Vac.
Diethyl procarbonate treatment: Stop the reaction by adding an equal volume
of DEPC stop buffer and phenol–CHCl
3
extract. Add 5 µL of 5 mg/mL
linear acrylamide, and precipitate with 2.5 volumes of ethanol as above.
After centrifugation, rinse with 70% ethanol and centrifuge again. Remove
supernatant and dry pellet on medium heat for 5 min in a Speed Vac.
5. Alkaline cleavage: Suspend the pellet in 50 µL of 1 M piperidine (10% v/v) and
incubate at 90°C for 30 min. Place a lead weight on top of the tubes or use tube
locks to prevent the lids from opening.
Use of DEPC and Potassium Permanganate as Probes 69
6. Place tubes on ice to cool and centrifuge briefly. Add 50 µL of 0.6 M sodium
acetate, 300 µg/mL linearized polyacrylamide, and 250 µL 95% ethanol. Mix
and centrifuge 30 min as above. Wash pellet with 150 µL of 70% ethanol. Spin
samples for 5 min. Remove supernatant.
7. To remove residual piperidine, add 30 µL sterile deionized water to each sample
and dry on medium heat in a Speed Vac (see Note 5).
8. Add 5 µL electrophoresis loading buffer, vortex samples for 30 s, and heat
samples at 95°C for 3 min. Place samples on ice.
9. Load samples on sequencing gel and analyze by standard methods (see Notes 6 and 7).
3.2. Treatment of DNA In Vivo with KMnO4 and Purification
Potassium permanganate has been used to modify DNA in vivo, followed
by analysis of modifications by primer extension or polymerase chain reaction
(PCR). DEPC cannot be used for in vivo experiments because of its low solu-
bility. The procedure for in vivo modification is fairly straightforward, how-
ever, certain nutrient-rich media can quench permanganate. To avoid this
problem, use minimal medium or increase the permanganate concentration so
that the reaction mixture does not turn brown in less than 1 min. For some
experiments, one can dilute the culture in minimal medium just prior to treat-
ment with potassium permanganate. In vivo modification of mammalian cell
cultures usually requires the removal of the growth medium just prior to treatment.
1. To 10 mL of diluted bacterial or yeast culture, add the appropriate amount of
KMnO
4
(typically in the low millimolar range, depending on the medium, approx
10–20 mM for most media) for the desired amount of time (10 s to 5 min) in a
shaking water bath. To quench treatment, remove samples from the water bath
and pour immediately into prechilled Corex tubes and add 2-mercaptoethanol
until the purple color disappears. Centrifuge to pellet cells in a cold Sorvall SS34
rotor for 5 min at 3015g. Discard the supernatant.
2. For mammalian cells grown to subconfluence on plastic growth dishes, remove
growth medium and wash twice with phosphate-buffered saline or minimal
growth medium. Add the desired concentration of potassium permanganate (usu-
ally 2–20 mM) for the necessary period of time (10 s to 5 min). Stop the perman-
ganate reaction by washing cell monolayers twice with phosphate-buffered saline
containing 2% 2-mercaptoethanol and once with phosphate-buffered saline. Cells
are then harvested with a rubber policeman or cell scraper.
3. Plasmid and genomic DNA can be isolated by a variety of standard methods
(49–51). Purified modified DNA should be adjusted to a final concentration of
approx 15 ng/µL and be free of contaminants that interfere with extension reac-
tions. Extractions involving phenol should be repeated three to four times or until
there is no contaminating interphase. Modifications to the DNA can then be visu-
alized by PCR amplification, as detailed in Subheading 3.4. An alternative
method for identifying modified genomic DNA is the use of ligation mediated
PCR (LMPCR) (52,53).
70 Kahl and Paule
3.3. Primer Extension Analysis of DNA Treated In Vitro or In Vivo
Because DNA modified in vivo or when in circular form is not end labeled,
primer extension is the method of choice to analyze the sites of modification.
Modified bases result in extension stop sites because they block the elongating
DNA polymerase. For in vitro studies, follow steps 2–4 of Subheading 3.1.,
substituting 20–500 ng of purified DNA in place of radiolabeled DNA. For in
vivo studies, follow the steps of Subheading 3.2.
1. To the isolated, modified DNA (20–500 ng for in vitro and 500 ng for in vivo
studies), add (0.3–0.5) × 10
6
cpm of 5' end-labeled primer and dilute to 36 µL
with distilled water.
2. Add 4 µL of 0.01 M NaOH to each reaction and mix well.
3. Denature DNA by heating to 95°C for 2 min.
4. Add 5 µL of 10X neutralization buffer and mix.
5. Hybridize primer to DNA by heating sample for 3 min at or just under the calcu-
lated T
m
of the primer.
6. Add 5 µL of a solution containing all four dNTPs at a concentration of 5 mM
each.
7. Add 0.5–1.0 unit of the Klenow fragment of DNA polymerase I and mix gently.
Incubate tube for exactly 10 min at 50°C.
8. Quench by adding an equal volume (~50 µL) of 0.6 M sodium acetate, and 20 mM
EDTA and place on ice.
9. Precipitate DNA by adding 300 µL of 95% ethanol, mix and centrifuge 30 min.
Wash the pellet with 150 µL of 70% ethanol. Centrifuge for 5 min, remove super-
natant, and dry pellet.
10. Suspend pellet in 5 µL electrophoresis loading buffer and run on a normal
sequencing gel. Analyze by standard techniques (see Notes 8 and 9).
3.4. PCR Amplification of DNA Treated In Vivo or In Vitro
1. In a 0.65-mL microcentrifuge tube, add the following:
5 µL 10X reaction buffer supplied with the thermostable polymerase
2 µL dNTP mix
0.5 × 10
6
cpm end-labeled primer
Distilled water to a final volume of 49.5 µL
2. Program thermocycler. For example:
1 Round:
2 min at 95°C , pause and add 0.5 µL of thermostable polymerase
(2.5 units/µL)
30 s at T
m
of primer
30 s at 72°C
15–20 Rounds:
1 m at 95°C
30 s at T
m
of primer
30 s at 72°C
Use of DEPC and Potassium Permanganate as Probes 71
1 Round:
5 min at 72°C
3. Precipitate DNA by adding 50 µL of 0.6M sodium acetate, 300 mg/mL linear
polyacrylamide, and 250 µL 95% ethanol. Mix and centrifuge 30 min at
14,000 rpm in a microfuge. Wash pellet with 150 µL of 70% ethanol and
centrifuge for 5 min. Remove supernatant and dry pellet on medium heat for
5 min in a Speed Vac.
4. Suspend pellet in 5 µL loading buffer and run on sequencing gel. Analyze by
standard techniques (see Notes 8 and 9).
4. Notes
1. False positive results can occur from the presence of nucleases in any of the
proteins being tested. A necessary control is to incubate each protein with the
DNA in the absence of further treatment with the modifying reagent. The DNA
isolated from these reactions is run through the remainder of the analysis proce-
dure to reveal any digestion of the DNA by contaminating nucleases.
2. Certain sequences of DNA are sensitive to DEPC and potassium permanganate
treatment even in the absence of proteins. It is important to run a control lane of
DNA to obtain a background level of sensitive sites.
3. To optimize reaction conditions, a titration of potassium permanganate for vary-
ing amounts of time may be necessary. Too little potassium permanganate results
in no signal, and too much potassium permanganate can result in a high back-
ground. Two to five millimolars of potassium permanganate is typical for in vitro
experiments, but for in vivo experiments, where the medium may quench the
reagent, concentrations up to 200 mM can be used. Times of reaction have been
varied from 10 s up to 5 min, but in our hands, there is much less difference in the
results obtained with different reaction times than with different potassium per-
manganate concentrations. Thus, one can set up the experiment for a convenient
period of time.
4. Potassium permanganate and DEPC react with proteins as well as DNA, which
can impair their function. Thus, negative experiments may result from protein
denaturation rather than a lack of DNA modification by the protein.
5. It is important to remove all the piperidine from the DNA following cleavage. If
smeared bands are found on the gel, try doing more than one round of drying in
the Speed Vac by redissolving the pellet in 30 µL of deionized water and drying
as described in step 7 of Subheading 3.1.
6. To obtain the maximum amount of information from the DNA of interest, perform
separate experiments with either the template or the RNA-like strand radiolabeled.
7. A Maxam and Gilbert sequencing ladder of the DNA being analyzed run adja-
cent to the probing reactions is useful to identify specific modified sites.
8. For primer extension and PCR amplification reactions, several factors can affect
the observed signal. A loss of signal can be the result of the following:
a. Improper primer sequence
b. Annealing temperature higher than T
m
72 Kahl and Paule
c. Contaminants present in reaction
d. High concentration of magnesium ion
9. Extra bands or smearing can occur if the annealing temperature is suboptimal and
allows mispriming. Nonspecific hybridization can also occur if the radiolabled
primer has undergone extensive decay. Freshly labeled primer reduces the risk of
mispriming events. Supercoiled DNA can cause sequence-induced stopping of
the DNA polymerase. Linearizing the plasmid before fill-in or amplification can
reduce improper extension.
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In Gel
OP–Cu Footprinting 77
77
From:
Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed.
Edited by: T. Moss © Humana Press Inc., Totowa, NJ
7
Footprinting DNA–Protein Interactions
in Native Polyacrylamide Gels by Chemical
Nucleolytic Activity of 1,10-Phenanthroline-Copper
Athanasios G. Papavassiliou
1. Introduction
The existence of cell-type specific promoter and enhancer elements has
been known for several years. However, the mechanisms responsible for the
remarkable specificity of such elements, in comparison to the ubiquitously
active promoters and enhancers of “housekeeping” genes and DNA tumor
viruses, have remained elusive until recently. Although transfection and
mutagenesis experiments have taught us a great deal about the structure of
cell-type-specific cis-acting elements, the breakthrough in understanding the
molecular basis for the differential activity of these elements has come from
the analysis of their recognition by specific DNA-binding proteins.
Several techniques have been developed for the detection of cell-type-spe-
cific DNA-binding activities and the identification of sequence-specific con-
tacts (“footprints”) of a protein on DNA. Many of these techniques involve
forming DNA–protein complexes (by incubating an asymmetrically labeled
double-stranded DNA fragment containing the region of interest with a crude
or partly purified protein extract), exposing the complex to enzymatic or to
chemical reagents that can cleave or modify the DNA, and determining which
bases are protected from attack when the protein(s) is bound. The most widely
used reagents are deoxyribonuclease I (DNase I, an endonuclease) and dim-
ethyl sulfate (DMS). Footprinting experiments with DNase I are performed
using parallel reactions with free DNA and with DNA–protein complexes, and
the nuclease is allowed to digest DNA only to a limited extent (1). The DNA is
then purified and denatured, and the single-stranded end-labeled DNA frag-
ments are resolved on a sequencing gel and autoradiographed. Comparing the