Moss Tom. DNA-protein interactions: principles and protocols
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DNase I Footprinting 35
The following protocol was developed to study the footprinting of the Xeno-
pus ribosomal transcription factor xUBF, which is a rather weak DNA-binding
protein with a rather broad sequence specificity. The protocol is not original,
being derived from several articles (e.g., refs. 1 and 7). It does however repre-
sent a very practical approach which can be broadly applied. We recommend
that the reader also refer to (6) for more information on the quantitative analy-
sis of protein–DNA interactions by footprinting.
2. Materials
1. Binding buffer (2X): 20% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol (DTT)
20 mM HEPES pH 7.9 and 4% poly(vinyl alcohol) (see Note 2).
2. poly dAdT–poly dAdT (Pharmacia LKB): 1 mg/mL solution in TE. Keep at –20°C
(see Note 3).
3. End-labeled DNA fragment of high-specific activity (see Note 1).
4. Cofactor solution: 10 mM MgCl
2
and 5 mM CaCl
2
.
5. DNase I stock solution: A standardized vial of DNase I (Sigma, D4263) is dis-
solved in 50% glycerol, 135 mM NaCl, 15 mM CH
3
COONa pH 6.5 at 10 Kunitz
units/µL. This stock solution can be kept at –20°C for many months (see Note 4).
6. 1 M KCl
7. Reaction Stop buffer: 1% sodium dodecyl sulfate (SDS), 200 mM NaCl, 20 mM
EDTA pH 8.0, and 40 µg/mL tRNA (see Note 5).
8. 10X TBE buffer: 900 mM Tris-borate pH 8.3, 20 mM EDTA
9. Loading buffer: 7 M urea, 0.1X TBE, 0.05% of xylene cyanol and of bromophe-
nol blue.
10. 6% acrylamide, 7 M urea, and 1X TBE sequencing gel.
11. Phenol–chloroform (1:1) saturated with 0.3 M TNE.
3. Methods
The footprinting reaction is done in three stages: binding of the protein to
the DNA, partial digestion of the protein–DNA complex with DNase I, and
separation of the digestion fragments on a DNA sequencing gel.
1. The binding reaction is performed in a total volume of 50 µL containing 25 µL of
2X binding buffer, 0.5 µL of 1 mg/mL poly dAdT. poly dAdT, 2–3 ng of end-
labeled DNA fragment (approx 15,000 CPM), (see Note 6), the protein fraction
and 1 M KCl to bring the final KCl concentration to 60 mM. The maximum vol-
ume of the protein fraction that can be used will depend on the salt concentration
of this solution. The reaction is performed in a 1.5-mL Eppendorf tube.
2. Incubate on ice for 20 min.
3. During the binding reaction, dilute the DNase I stock solution in water at 0°C.
We suggest working concentrations of about 0.0005 to 0.1 Kunitz units/µL,
depending on the level of protein present (see Note 7 and step 5). A good range
is 0.0005; 0.001; 0.002; 0.005; 0.02; 0.08 U/µL.
36 Leblanc and Moss
4. After the incubation, transfer the reaction tubes in batches of eight to a rack at
room temperature (RT) and add 50 µL of the cofactor solution to each.
5. Add 5 µL of the appropriate DNase I dilution to a tube every 15 s. (0.0005–0.005
for naked DNA; 0.002–0.08 for DNA + proteins).
6. After 2 min digestion, each reaction is stopped by the addition of 100 µL of the
stop solution (RT), see Note 8.
7. After all the reactions have been processed, phenol–chloroform extract each
reaction once.
8. Add two volumes (400 µL) of ethanol (–20°C) and allow nucleic acids to precipi-
tate at –80°C for 20 min.
9. Microcentrifuge for 15 min, at approx 10,000g and remove the supernatant with
a Pasteur pipet. Check for the presence of a radioactive pellet before discarding
the ethanol.
10. Add 200 µL of 80% ethanol (–20°C) and microcentrifuge again. After removing
supernatant, dry the pellets in a vacuum desiccator.
11. Resuspend each pellet in 4.5 µL loading buffer, vortex, and centrifuge briefly.
12. A G+A ladder and a molecular-weight marker should be run in parallel with the
samples on the sequencing gel (step 13) (see Note 9). The G+A ladder can be
prepared as follows (5): approx 200,000 cpm of end-labeled DNA are diluted
into 30 µL H
2
O (no EDTA). 2 µL of 1 M piperidine formate, pH 2.0, are added
and the solution incubated at 37°C for 15 min; 150 µL of 1 M piperidine are
added directly and the solution incubated at 90°C for 30 min in a well-sealed tube
(we use a 500-µL microtube in a thermal cycler). Add 20 µL of 3 M CH
3
COONa
and 500 µL of ethanol and precipitate at –80°C for 10–20 min. Micro-
centrifuge (10,000g, 10 min) and repeat precipitation. Finally, redissolve in 200 µL
of H
2
O and lyophilize. Resuspend in gel loading buffer and apply about 5000 cpm
per track.
13. Prerun a standard 6% acrylamide sequencing gel for 30 min before loading each
of the aliquots. Wash the wells thoroughly with a syringe, denature the DNA for
2 min at 90°C, and load with thin-ended micropipet tips. Run the gel hot to keep
the DNA denatured (see Note 10). After the run, wrap the gel in plastic wrap and
expose it O/N at –70°C with an intensifying screen (see Note 11). Several differ-
ent exposures will probably be required to obtain suitable band densities.
4. Notes
1. Single-stranded breaks in the end-labeled DNA fragment must be avoided as they
give false signals indistinguishable from genuine DNase I cleavage and hence
can mask an otherwise good footprint. It is therefore advisable to check the
fragment on a denaturing gel before use. Always use a freshly labeled fragment
(3–4 d at the most), as radiochemical nicking will degrade it.
2. This binding buffer has been shown to work well for the transcription factor NF-1
(6) and in our lab for both the hUBF and xUBF factors and thus should work for
many factors. Glycerol and poly(vinyl alcohol) (an agent used to reduce the avail-
able water volume and hence concentrate the binding activity) are not manda-
DNase I Footprinting 37
tory. The original footprinting conditions of Galas and Schmitz (1) for the bind-
ing of the lac repressor on the lac operator were 10 mM cacodylate buffer pH 8.0,
10 mM MgCl
2
, 5 mM CaCl
2
and 0.1 mM DTT. Particular conditions of pH,
cofactors and ionic strength may need to be determined for an optimal binding of
different factors to DNA.
3. Because poly dIdC, another nonspecific general competitor, has been shown to
compete quite efficiently with G–C-rich DNA sequences, poly dAdT is preferred
here. The choice of an appropriate nonspecific competitor (be it synthetic as in
this case or natural, for example, pBR322 or calf thymus DNA) may have to be
determined empirically for the protein studied. When working with a pure or
highly enriched protein, no competitor is usually needed. The DNase I concen-
tration must then be reduced accordingly (to about naked DNA values).
4. These standardized vials allow for very reproducible results. Glycerol will keep
the enzyme from freezing, as repeated freeze–thaw cycles will greatly reduce
its activity.
5. Do not be tempted to use too much RNA, as it causes a very annoying fuzziness
of the gel bands, preventing resolution of the individual bands. The RNA carrier
can be completely omitted if care is taken at the precipitation step.
6. The use of 5' end labeling with polynucleotide kinase in the presence of crude
protein extracts can sometimes lead to a severe loss of signal because of the pres-
ence of phosphatases. In these cases 3' end labeling by “fill out” with Klenow or
T
4
DNA polymerases is to be preferred.
7. For naked DNA and very low amounts of protein, dilutions of 0.0005 to 0.005
give a good range of digestion.
8. It is convenient to work with groups of eight sample during the DNase I digestion.
The cofactor solution is added to eight samples at a time and then the DNase I
digestions are begun at 15-s intervals. Fifteen seconds after adding DNase to the
eighth sample, the stop solution is added to sample 1 and then to the other samples
at 15-s intervals.
9. In comparing a chemical sequencing ladder with the products of DNase I diges-
tion, one must bear in mind that because the chemical modification and cleavage
destroys the target base, each band in the sequencing ladder corresponds to a
fragment ending in the base preceding the one read. For example, if a DNase I gel
band corresponds in mobility to the sequence ladder band read as G in the
sequence ACGT, then the DNase I cleavage occurred between the bases C and G.
DNase I cleaves the phosphodiester bond, leaving a 3'-OH, whereas the G+A and
C+T sequencing reactions leave a 3'-PO
4
, causing a mobility shift between the
two types of cleavage ladders. This is a further potential source of error. How-
ever, in our experience, the shift is less than a half a base and, hence, cannot lead
to an error in the deduced cleavage site.
10. Sequencing gels are not denaturing unless run hot (7 M urea produces only a
small reduction in the T
m
of the DNA). A double-stranded form of the DNA
fragment is therefore often seen on the autoradiogram, especially at low levels of
DNase I digestion and can sometimes be misinterpreted as a hypersensitive cleav-
38 Leblanc and Moss
age. By running a small quantity of undigested DNA fragment in parallel with
the footprint, this error can be avoided.
11. For detection of
32
P-labeled DNA in sequencing gels we have used either Cronex
Lightning Plus (Dupont) or Kyokko Special (Fuji) intensifying screens, the latter
being, in practice, 30% less sensitive but often much less expensive. Fuji-RX or
similar films are, in practice, 30% slower than Kodak X-omat AR film, but in
North America, at the time of writing, they are five to six times less expensive.
Hence, the combinations of screens and films Kyokko/RX:Cronex/RX: Kyokko/
AR:Cronex/AR give relative sensitivities of about; 1:1.5:1.5:2. Should the newer
slightly higher sensitivity films be used (e.g., Kodak BioMax), it is essential that
the appropriate intensifying screens be employed. These newer films are usually
most sensitive to green light and do not work well with the Cronex Lightning
Plus/Kyokko Special type screens, which emit a blue light.
Acknowledgments
The authors wish to thank Dr. A. Ruiz-Carrillo for providing the autoradio-
gram in Fig. 1A. This work was supported by a project grant from the Medical
Research Council (MRC) of Canada. Tom Moss is an MRC of Canada Scien-
tist and a member of the Centre de Recherche en Cancérologie de l’Université
Laval which is supported by the FRSQ of Québec.
References
1. Schmitz, A. and Galas, D. J. (1978). DNAase I footprinting: a simple method
for the detection of protein-DNA binding specificity. Nucleic Acids Res. 5,
3157–3170.
2. Rousseau, S., Renaud, J., and Ruiz-Carrillo, A. (1989). Basal expression of the
histone H5 gene is controlled by positive and negative cis-acting sequences.
Nucleic Acids Res. 17, 7495–7511.
3. Suck, D., Lalm, A., and Oefner, C. (1988). Structure refined to 2 Å of a nicked
DNA octanucleotide complex with DNAse I. Nature 332, 464–468.
4. Drew, H. R. (1984). Structural specificities of five commonly used DNA
nucleases. J. Mol. Biol. 176, 535–557.
5. Maxam, A. M. and Gilbert, W. (1980). Sequencing end-labeled DNA with base-
specific chemical cleavages, in Methods in Enzymology, Vol. 65 (Grossman, L.
and Moldave, K., eds.), Academic, New York, pp. 499–560.
6. Brown, T. (1987). Analysis of RNA by Northern and slot blot hybridisation, in
Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R.
E., Moore, D. D., Seidman, J. G., Smith, J. A., et al., eds.), Greene Publishing
Associates/Wiley-Interscience, New York, pp. 4.9.1–4.9.14.
7. Walker, P. and Reeder, R. H. (1988). The Xenopus laevis ribosomal gene
promoter contains a binding site for nuclear factor-1. Nucleic Acids Res. 16,
10,657–10,668.
Footprinting with Exonuclease III 39
39
From:
Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed.
Edited by: T. Moss © Humana Press Inc., Totowa, NJ
4
Footprinting with Exonuclease III
Willi Metzger and Hermann Heumann
1. Introduction
Within the last few years footprinting techniques have become increasingly
important in the study of protein–nucleic acid interactions. This is partly the
result of a fast-growing number of known nucleic acid-binding proteins but
also because of an increase in the available probes that can be chosen in order
to tackle a specific problem. There are two major groups of probes—the chemi-
cal probes and the enzymatic probes. The enzymatic probes, such as DNase I
or exonuclease III, have the advantage of acting specifically on the DNA.
Chemical probes are often less specific and may also react with the protein,
possibly disturbing the correct interaction of protein with DNA. For the study
of very fragile protein–DNA complexes, enzymatic probes are therefore often
preferable.
The exploitation of a specific enzymatic function can also be a reason for
choosing an enzymatic probe. The exonuclease activity and processivity of
exonuclease III makes this enzyme a suitable probe when information about
the position of a sequence-specific bound protein is required. A prerequisite
for the successful use of exonuclease III as a footprinting probe is, however,
that the half-life of the protein–DNA complex should be long compared with
the time required for the exonuclease III reaction.
1.1. Enzymatic Activities of Exonuclease III
Exonuclease III (Exo III) is a monomeric enzyme with a molecular weight
of 28,000 kDa. It contains several distinct activities: a 3'–5' exonuclease activ-
ity, a DNA 3' phosphatase activity, an AP endonuclease activity, and an RNase
H activity (1).
40 Metzger and Heumann
1.2. Principle of the Procedure
Footprinting with Exo III makes use of the 3'–5' exonuclease activity of
this enzyme (2). After a protein has been specifically bound to a DNA frag-
ment containing its recognition site, Exo III is used to remove mononucle-
otides from both DNA strands in a processive way, beginning from the 3'
termini. The specifically bound protein blocks the action of Exo III and
leaves double-stranded DNA only in the region bound by the protein (Fig. 1).
(Any free DNA is fully digested, an advantage of Exo III over other
footprinting probes, as there are no background problems caused by the
presence of free DNA.) The lengths of the two resultant protected single-
strand DNA fragments are determined by electrophoresis on a denaturing
sequencing gel using appropriate DNA length standards, the fragments
being detected by autoradiography via a 5' radioactive end label. If both
termini of the initial DNA duplex are labeled, a decisive association of the
protected fragments with the upper and lower strands may be difficult. This
problem is overcome by removal of one end-label (e.g., by a restriction
enzyme cleavage before Exo III digestion). Figure 1A shows the procedure
schematically and Fig. 1B shows the expected radioactive products (bands)
after denaturing gel electrophoresis.
1.3. Interpretation of Results
The region of a DNA duplex protected by a bound protein can be deter-
mined from the length of the two single-strand DNA fragments remaining after
Exo III treatment. If the initial DNA duplex has a length of k base pairs and the
single-strand products have lengths of m and n bases, respectively, the size of
the protected region x is given by;
x = m + n – k
Correct interpretation of the footprinting data, however, requires a critical
assessment of the action of the Exo III on the protein–DNA complex. The
interpretation is straightforward if the protein is strongly bound to DNA. In
this case, because the protein acts as a steric hindrance for Exo III, the protected
region gives an upper limit of the size of the DNA segment interacting with the
protein. Interpretation of the data is more complicated if the strength of
interaction between protein and DNA varies within the interacting domain.
Because of the processivity of Exo III, this enzyme may “nibble” into a pro-
tein–bound DNA segment for which the binding protein has lower affinity,
resulting in an underestimation of the extent of protein–DNA contact. In order
to decide if such a process occurs, it is essential to study a time-course of the
Exo III digestion.
Footprinting with Exonuclease III 41
1.4. Examples of the Application of Exo III
as a Footprinting Probe
Exonuclease III has been used to follow the movement of E. coli RNA poly-
merase during RNA synthesis (3,4). RNA synthesis was arrested at specific
positions and the arrested complexes were subjected to Exo III digestion. A set
of single–strand products marking the boundaries of RNA polymerase on the
DNA at each step of RNA synthesis was observed. Exo III also offers the pos-
sibility of detecting specific DNA–protein interactions in crude extracts,
because those proteins whose half-lives are greater than the reaction time act
as a block for Exo III (5–7).
Fig. 1. (A) Schematic representation of the Exo III footprinting procedure. (B) Exo
III footprint of the complex shown in (A). Lane 1: Size markers; lane 2: labeled DNA
fragment; lane 3: labeled DNA fragment, after Exo III treatment; lane 4: DNA from
DNA–protein complex, after Exo III treatment, both 5' ends of duplex labeled; lane 5:
DNA from DNA–protein complex, after Exo III treatment, DNA duplex singly end-
labeled label was removed beforehand by cutting with a restriction enzyme as indi-
cated in (A).
42 Metzger and Heumann
2. Materials
2.1. Exonuclease III
Exonuclease III is available from BRL (Gaithersburg, MD) or Boehringer
Mannheim (Indianapolis, IN). It can be stored for many months at –20°C.
2.2. Sequencing Gel
1. Acrylamide solution: 40% acrylamide, and 0.66% bis-acrylamide in H
2
O.
2. 10X TBE: 1 M Tris-HCl, pH 8.6, 840 mM boric acid; and 10 mM EDTA.
3. 10% Ammonium persulfate (freshly prepare before use).
4. N,N,N'N'-tetramethylethylene diame (TEMED).
5. Preparation of an 8% acrylamide gel: Weigh 21 g of urea, and add 5 mL of 10X
TBE solution and 10 mL of acrylamide solution. Dissolve under mild heating.
Add double-distilled water to a final volume of 50 mL. Filter and degas the solu-
tion (filter pore size 0.2 µm). Add 500 µL of 10% ammonium persulfate solution
and 30 µL of TEMED. Pour gel immediately.
6. Formamide loading buffer for the sequencing gel: 100 mL deionized formamide,
30 mg xylenecyanol FF, 30 mg bromophenol blue, and 750 mg EDTA.
7. Electrophoresis buffer: 1X TBE.
2.3. Nondenaturing Gel for the Band-Shift Assay
1. Acrylamide solution: 30% acrylamide and 0.8% bis-acrylamide in H
2
O.
2. 1 M Tris-HCl, pH 7.9.
3. 10% Ammonium persulfate (freshly prepare before use).
4. 5% TEMED (diluted in water).
5. Preparation of nondenaturing gel: Mix 240 µL of 1 M Tris-HCl, pH 7.9, 2.75 mL
acrylamide solution, and 25.7 mL of H
2
O and degas. Add 300 µL of 10% ammo-
nium persulfate and 70 µL of 5% TEMED. Pour gel.
6. Loading buffer for the nondenaturing gel (10X solution): 40% sucrose and 0.1%
bromophenol blue.
7. 5% Dichloro-dimethylsilane solution (in chloroform).
8. 0.3% g-Methacryl-oxypropyl-trimethoxy-silane and 0.3% acetic acid dissolved
in ethanol.
9. Electrophoresis buffer: 8 mM Tris-HCl, pH 7.9.
Store the solutions protected from light at 4°C. Dilute buffers with
bidistilled water.
2.4. Other Items
1. Sequencing gel apparatus (Pharmacia, Piscataway, NJ).
2. Filters for drop dialysis VS, 0.025 µm (Millipore, Bedford, MA).
3. Peristaltic pump.
4. SpeedVac concentrator.
Footprinting with Exonuclease III 43
3. Methods
3.1. Establishing Conditions for Optimum Yield
of Specific Protein–DNA Complexes
A very elegant method for establishing the optimum conditions for the
formation of a specific protein–DNA complex is acrylamide gel electrophore-
sis under nondenaturing conditions. This electrophoretic mobility shift assay
(EMSA) or “band shift assay” allows one to differentiate between complexed
and uncomplexed DNA (8,9) and thus to determine the stoichiometry of given
complexes and the optimum salt conditions for their formation (see Chapter 2).
This method can even be applied to high-molecular-weight protein DNA
complexes, given the acrylamide concentration is low enough to enable the
complex to enter the gel matrix. The gel composition given in Subheading
2.3. is optimal for the study of high-molecular-weight complexes (see Note 6).
To facilitate the handling of low-concentration polyacrylamide gels, the
glass gel plates are subjected to a special treatment ensuring the gel will remain
bound to only one of the two plates after electrophoresis:
1. Wash the glass plates (20 × 20 cm) with ethanol.
2. Treat one plate with g-methacryl-oxypropyl-trimethoxysilane solution, then wash
this plate carefully four times with ethanol.
3. Treat the second plate with dichloro-dimethylsilane solution.
4. Form the protein–DNA complex in a volume of about 15 µL under the desired
conditions.
5. Dialyze the complex, if necessary, against the electrophoresis buffer by drop
dialysis as follows:
a. Pour the dialysis buffer into a Petri dish.
b. Place a VS filter (Millipore, see Subheading 2.4.) with the glossy side upward
onto the buffer so that it can float freely.
c. Place the sample as a drop on the filter. Remove the drop after 1 h when
dialysis is complete.
6. Add 1/10 volume of the 10X loading buffer to the dialyzed complex and apply
the sample to the nondenaturing polyacrylamide gel.
7. Run the gel at 20 V/cm for approx 2 h. Pump the buffer from the anode to the cathode
chambers and back again to avoid pH decrease in the anode chamber (see Note 1).
3.2. Establishing the Conditions for the Digestion of the DNA
1. Label the DNA at the 5'-ends using T
4
polynucleotide kinase and [γ-
32
P] ATP.
Take an aliquot of end-labeled DNA and remove one 5'-label by asymmetric
cleavage with an appropriate restriction enzyme. Use the cleaved and uncleaved
labeled DNA in separate analyses as described the following steps.
2. Ensure that the total amount of DNA in an assay of 20 µL is not below 100 ng.
The total amount of radioactivity in one assay should be approx 20,000 cpm. Use
44 Metzger and Heumann
the optimal salt conditions optimal for complex. Add 6 mM Mg
2+
, if this is not
already present.
3. Add Exo III and incubate at 37
o
C. In order to establish the optimum conditions,
perform a series of experiments using concentrations Exo III between 1 and
200 U per reaction (20 µL) and incubation times varying between 1 and 45 min.
Exo III seems to be rather stable over a wide range of ionic strengths, and no
large changes in activity were observed in the range between 0 and 100 mM NaCl
(or KCl) in the incubation mixture (see Note 2).
4. Add EDTA to a final concentration of 20 mM in order to stop the reaction at the
appropriate time.
5. Add sodium acetate to a final concentration of 0.3 M followed by 2.5 volumes of
ice-cold 100% ethanol to precipitate the digested DNA. Keep the solution at –70°C
for 20 min.
6. Spin down the solution in a microcentrifuge for 15 min. Wash the pellet with
ice-cold 75% ethanol, dry under vacuum, and dissolve in formamide load-
ing buffer.
7. Heat sample at 100
o
C for 2 min and apply onto a 6–10% sequencing gel. (For the
analysis of fragments in the range of 50–150 bases, 8% polyacrylamide is
adequate, as described in Subheading 2.2.). Use as a length standard a Maxam–
Gilbert sequencing reaction of the 5'-end-labeled DNA fragment.
8. Run the gel at 50 W and a temperature of 60°C for 2 h.
9. After electrophoresis, expose the gel overnight at –70°C to X-ray film using an
intensifying screen.
10. Ensure that the free DNA is fully digested (usually much shorter single-strand
DNA fragments than the predicted half-full-length [2] are obtained).
3.3. Exo III Digestion of the Protein–DNA Complex
1. Form the complex using the conditions established under Subheading 3.1.
2. Subject the complex to Exo III digestion using the conditions established under
Subheading 3.2. (see Notes 1 and 3–6).
3. Add 20 mM EDTA, 1% sodium dodecyl fulfate (SDS) (final concentration) to
stop the reaction. SDS is necessary in order to destroy the protein–DNA complex.
4. Proceed as described in Subheading 3.2. (steps 5–9) for recovery and gel elec-
trophoretic analysis of the DNA. For recovery of the DNA, a phenol extraction
before the ethanol precipitation is advisable if a crude protein extract has been
used for complex formation.
3.4. Modifications of the Procedure
Depending on the kind of protein–DNA complexes being investigated, sev-
eral modifications of the procedure described in the previous section may be
useful or necessary. If the protein–DNA complexes are not homogeneous (e.g.,
more than one type of protein complex can form or the DNA contains multiple
binding sites for a given protein), the desired complex can be purified on a