Moss Tom. DNA-protein interactions: principles and protocols
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Ethylation Interference 233
5. Solutions for ethanol precipitation of DNA: 4 M NaCl and ethanol (absolute and
70% v/v).
6. Alkaline phosphatase (AP), from calf intestine (CIAP) or shrimp.
7. 10X AP reaction buffer: 0.5 M Tris-HCl, pH 9.0, 0.1 M MgCl
2
, and 0.001 M ZnCl
2
.
8. TE buffer: 10 mM Tris-HCl, pH 8.0, and 1 mM ethylenediaminetetraacetic acid
(EDTA).
9. Sodium dodecyl sulfate (SDS) 20% w/v.
10. EDTA (0.25 M, pH 8.0).
11. T4 polynucleotide kinase (T4-PNK).
12. 10X T4-PNK reaction buffer: 0.5 M Tris-HCl, pH 7.6, 0.1 M MgCl
2
, and 0.05 M
dithiothreitol (DTT).
13. Radioisotope: [γ-
32
P]-ATP.
14. 30% w/v Acrylamide stock: 29:1 :: acrylamide: N,N'-methylene–bis-acrylamide.
15. Polyacrylamide gel elution buffer: 0.3 M sodium acetate, 0.2% (w/v) SDS,
and 2 mM EDTA.
16. Polymerization catalysts: ammonium persulfate (APS) (10% w/v) and N,N,N',N'-
tetramethylethylenediamine (TEMED).
2.2. Ethylation Modification and Fractionation of DNA
1. End-labeled DNA in TE: 250,000 cpm are required per ethylation reaction to be
performed (roughly 20 ng DNA fragment/reaction). Ethylation of more DNA
allows a range of protein concentrations to be used when protein–DNA binding
reactions are prepared. Standard safety procedures should be used when handling
radiolabeled DNA (e.g., work behind Lucite shields).
2. Cacodylate buffer: 50 mM sodium cacodylate (used without adjustment of the
pH, which is usually close to 8.0). Cacodylate buffer contains arsenic and there-
fore should be handled with caution. It is only necessary to prepare small vol-
umes (approx 10 mL) of this solution. Passing the solution through a 0.45-µm
filter is the preferred method of sterilization.
3. Ethylnitrosourea (EtNU): This reagent is readily synthesized (9) provided, the
appropriate safety measures are used. Commercial suppliers do not appear to
provide this material at present. The solid material should be stored at –20°C and
allowed to warm to room temperature before use. EtNU should be handled in a
fume hood and contaminated waste stored there until disposal. Wear two pairs of
latex gloves when handling samples containing EtNU.
4. tRNA (1 mg/mL).
5. Solutions for ethanol precipitation of DNA: 4 M NaCl and ethanol (absolute and
70% v/v).
6. Complex buffer: for MetJ, for example, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl,
and 1 mMS-adenosyl methionine p-toluene sulfonate salt (SAM). Add glycerol
to this buffer to 10% (v/v).
7. Purified DNA-binding protein or protein extract.
8. Nondenaturing gel acrylamide stock solution: 30% (w/v) (37:1 acrylamide:
N,N'-methylene–bis-acrylamide).
234 Manfield and Stockley
9. Electrophoresis buffer stock solution: 1.0 M Tris-HCl, pH 8.0, and solid SAM.
10. Ammonium persulfate (APS) (10% w/v).
11. TEMED.
12. Glass plates: 150 × 150 × 1.5 mm.
13. Peristaltic pump capable of recirculating buffer at 5–10 mL/min.
14. X-ray film.
15. Autoradiography cassette.
16. X-ray film developer and fixer.
17. Plastic wrap.
18. Scalpel blade.
19. Syringe needle.
20. Polyacrylamide gel elution buffer: 0.3 M sodium acetate, 0.1% (w/v) SDS,
and 1 mM EDTA.
21. 10 mM sodium phosphate, pH 7.0, and 1 mM EDTA.
22. 1.0 M NaOH (freshly prepared).
23. Acetic acid solutions: 1.0 M and 10% v/v.
24. Sequencing gel loading buffer: 80% (v/v) formamide, 0.5X TBE, 0.1% (w/v)
xylene cyanol, 0.1% (w/v) bromophenol blue.
25. Acrylamide stock solutions for sequencing gel: 19% (w/v) acrylamide, 1% (w/v)
N,N'-methylene–bis-acrylamide, and 50% (w/v) urea in TBE.
26. TBE (1X): 89 mM Tris, 89 mM boric acid, and 10 mM EDTA, pH 8.3.
2.3. Maxam–Gilbert Chemical DNA Sequencing Reactions
There is insufficient space to cover these methods in detail here, but exten-
sive information, materials, methods, and troubleshooting guides are readily
available in published literature (10,11).
3. Methods
3.1. Preparation of End-Labeled DNA
1. Digest the plasmid (e.g., 10 µg of the plasmid in a reaction volume of 200 µL
with one of the pair of restriction enzymes used to release a suitably sized
DNA fragment [usually <200 bp]). Extract the digest with an equal volume of
buffered phenol and then add 2.5 vol of ethanol to the aqueous layer to precipi-
tate the DNA.
2. Add 50 µL 1X AP reaction buffer to the ethanol-precipitated DNA pellet (<50 µg).
Add 1 U AP, incubate at 37°C for 30 min, followed by addition of a further
aliquot of enzyme, and incubate for a further 30 min. Terminate the reaction by
adding SDS and EDTA to 0.1% (w/v) and 20 mM, respectively, in a final volume
of 200 µL and incubate at 65°C for 15 min. Extract the digest with an equal
volume of buffered phenol, then with 1:1 phenol:chloroform, and finally ethanol
precipitate the DNA from the aqueous phase by addition of 2.5 vol of ethanol.
3. Redissolve the DNA pellet (~2.5 µg) in 18 µL 1X T4-PNK buffer. Add 20 µCi
[γ–
32
P]-ATP and 10 U T4-PNK, and incubate at 37°C for 30 min. Terminate the
Ethylation Interference 235
reaction by phenol extraction (followed by ethanol precipitation and a second
restriction enzyme digest) or by addition of nondenaturing gel loading buffer,
and electrophoresis on a nondenaturing polyacrylamide gel. We use 12% (w/v)
polyacrylamide gels (19:1, acrylamide:bis-acrylamide) with 1X TBE as the elec-
trophoresis buffer.
4. After electrophoresis, locate the required DNA fragments by autoradiography of
the wet gel. Excise slices of the gel containing the bands of interest using the
autoradiograph as a guide. Elute the DNA into 500 µL elution buffer overnight
(at least) at 37°C. Ethanol precipitate the DNA, wash the pellet thoroughly with
70% (v/v) ethanol, dry under vacuum, and rehydrate in a small volume of TE
buffer (e.g., 50 µL). Determine the radioactivity of the sample by liquid scintilla-
tion counting of a 1 µL aliquot.
3.2. Ethylation Modification and Protein-Binding Assay
3.2.1. Ethylation Reaction
1. Dispense the required volume containing approx 250,000 cpm of radiolabeled
DNA solution into an Eppendorf tube, add cacodylate buffer to a final volume of
100 µL and heat the solution to 50°C in a heating block. Prepare the minimum
volume of EtNU-saturated ethanol (at 50°C) for the required reactions, add 100 µL
of this to the DNA, mix, and incubate at 50°C for 60 min (see Notes 1 and 2).
2. Add to the sample 5 µL of 4 M NaCl, 2 µg of tRNA, and 150 µL of ethanol. Mix
and place at –20°C for 60 min or in a dry ice/ethanol bath for 15 min. Pellet the
DNA by centrifugation in a microfuge for 15 min and remove the supernatant
(store separately to be destroyed by incineration). Add 500 µL of 70% ethanol,
mix thoroughly, recentrifuge, and remove the supernatant. Dry the pellet briefly
under vacuum.
3.2.2. Fractionation of DNA by Gel Retardation Assay
The following procedure allows the separation of free and bound DNA by
means of gel retardation. The precise conditions will depend on the protein
under investigation (see Notes 3–6).
1. Mix 10 mL of nondenaturing acrylamide stock solution, 1.5 mL of 1.0 M
Tris-HCl, pH 8.0, 29.5 mL of distilled water, 0.2 mL of APS, 15 µL of TEMED,
and 1.5 mg of SAM, and pour into gel frame. Insert the well former and leave to
polymerize for 1–2 h.
2. When polymerized, insert the gel into the tank and connect peristaltic pump tub-
ing such that buffer is pumped in both directions (i.e., top to bottom and bottom
to top reservoirs). Pre-electrophorese gel for 30 min at 100 V.
3. Redissolve the pellet of ethylated DNA in complex buffer plus glycerol (see
Note 6). Set aside 10% of the ethylated DNA sample, which will be used as an
unfractionated control to indicate the variation in level of modification at each
residue. Dry this sample under vacuum and store at –20°C until step 1 of Sub-
heading 3.2.4. Add DNA-binding protein to a concentration that would saturate
236 Manfield and Stockley
unmodified DNA and incubate at 37°C for 15 min to allow complex formation.
Note: Exact conditions will vary depending on the protein being studied.
4. Load the DNA–protein complex solution onto the gel and electrophorese into the
gel at 250 V for 2 min. Reduce the voltage to 100 V and continue electrophoresis
until a small amount of bromophenol blue dye loaded into an unused lane has
reached the bottom of the gel.
3.2.3. DNA Recovery
1. After electrophoresis, remove one plate and wrap the gel and remaining plate in
clear plastic wrap film (we use Saran Wrap™). Cut a piece of X-ray film large
enough to cover the lanes used on the gel and fix it to the gel firmly with masking
tape. Using a syringe needle, make a number of holes through the film and gel
that will serve to orient the developed film with respect to the gel. Alternatively,
align the film and the gel using fluorescent marker strips.
2. Place the assembly in an autoradiography cassette and leave at 4°C overnight.
3. Take the film off the gel and develop as usual. When dry, align the film and gel
using the holes created previously. Using a syringe needle, make a series of holes
into the gel around the fragments of interest using the bands on the film as a
guide. Remove the film and excise the marked regions of polyacrylamide. Place
gel fragments (10 × 5 mm) in Eppendorf tubes, add 600 µL of gel elution buffer,
and incubate at 37°C overnight (at least).
4. Transfer 400 µL of the eluate to a fresh tube, add 2 µg of tRNA and 1 mL of
ethanol, mix, and place at –20°C for 60 min. Pellet DNA by centrifugation in a
microfuge for 15 min. Discard the supernatant (check for absence of radioactiv-
ity), wash the pellet with 500 µL of 70% ethanol, recentrifuge, and discard the
supernatant. Dry the pellet briefly under vacuum. If the DNA does not pellet
readily, incubate the sample at –20°C for longer or recentrifuge at 4°C.
3.2.4. Phosphotriester Cleavage and DNA Sequencing
1. Redissolve each pellet in 15 µL sodium phosphate buffer and add 2.5 µL of 1 M
NaOH. Seal tube with plastic film (e.g., Parafilm) and incubate at 90°C for
30 min. Centrifuge samples briefly to collect any condensation. Add 2.5 µL
of 1.0 M acetic acid, 2 µL of 4 M NaCl, 1 µg of tRNA, and 70 µL of ethanol.
Leave samples at –20°C for 60 min. Pellet DNA as described in step 4 of Sub-
heading 3.2.3.
2. Redissolve the pellet in 4 µL of sequencing gel loading buffer. Heat to 90°C for
2 min and load samples onto a 12% w/v polyacrylamide sequencing gel along-
side Maxam–Gilbert sequencing reaction markers. Electrophorese at a voltage
that will warm the plates to around 50°C. After electrophoresis, fix the gel in
1 L of 10% (v/v) acetic acid for 15 min. Transfer the gel to 3MM paper and
dry under vacuum at 80°C for 60 min. Autoradiograph the gel at –70°C with an
intensifying screen.
3. Compare lanes corresponding to bound, free, and control DNA for differences in
intensity of bands at each position. A dark band in the “free fraction” (and a
Ethylation Interference 237
corresponding reduction in the intensity of the band in the “bound fraction”)
indicates a site where ethylation interferes with complex formation. This is inter-
preted as meaning that this residue is contacted by the protein or a portion of the
protein comes close to the DNA at this point. For 5' end-labeled DNA, the
ethylation reaction products migrate slightly more slowly than the Maxam–
Gilbert chemical sequencing products (see Notes 7 and 8, and also Fig. 1).
3.3. Results and Discussion
The result of an ethylation interference experiment with MetJ is shown in
Fig. 2 along with densitometer traces showing quantitative comparisons of the
distribution of products in bound and free fractions (Fig. 3) (13). Visual
examination of the autoradiograph shows that ethylation at 5'-pG2 results in
total exclusion of such fragments from protein–DNA complexes. Densitom-
etry indicates that ethylation at other sites inhibits complex formation to vary-
ing degrees and that there are more sites in the 3' half-site than in the 5' half-site
for which ethylation inhibits complex formation. These data can be interpreted
in terms of the MetJ-DNA crystal structure (8). The site at which ethylation
completely inhibits complex formation corresponds to the phosphate 5' to the
guanine at position 2 of each met box. The crystal structure shows a contact to
this phosphate from the N-terminus of the repressor B-helix. Indeed, at the
center of the operator site, sequence-dependent distortions of the oligonucle-
otide fragment away from B-DNA result in displacement of this 5' G2 phos-
phate by up to 2 Å in the direction of the protein. Thus, this site of complete
inhibition of complex formation corresponds to an important contact between
the DNA and a secondary structural element in the protein, which presumably
is unable to adjust to the presence of a bulky ethyl group. The other sites of
ethylation interference effects are contacted by amino acid side chains and pep-
tide backbone groups in extended loops of the repressor. It might be expected
that side chains and loops could be flexible enough to reorient in order to reduce
steric hindrance between the protein and the ethyl group, thus explaining the
partial interference effects. Similar good correlations between the contacts
identified by ethylation interference experiments and those seen in crystals
have been demonstrated in other systems, such as the phage 434 repressor (14)
and the phage Lambda repressor (15).
MetJ shows a high level of sequence specificity; therefore, clear interfer-
ence effects are observed. However, we have observed that proteins with low-
sequence specificity show weak interference effects. Densitometry and
quantitation may be necessary to resolve these effects from the background of
cleavage from fragments shifted by nonspecifically bound proteins. Addition-
ally, proteins binding to DNA asymmetrically (e.g., to sites which do not show
the dyad symmetry common to prokaryotic repressor proteins) will show dif-
238 Manfield and Stockley
Fig. 3. Summary of the results. Top: Densitometer traces of ethylated DNA from
the bound and free fractions. DNA from the free fraction is indicated by the trace with
solid lines and DNA from the bound fraction by the trace with dashed lines. The posi-
tion of each binding site is indicated at the bottom of the trace. The sequence within
each box is 5'-AGACGTCT–3'. Note that the greatest inhibitory effect is at the second
residue (G2) of each box and that the number of positions, ethylation of which inhibits
complex formation, is greater in the 3' box than in the 5' box. Bottom: Space filling
representation of the operator site showing the positions where ethylation results in
complete inhibition of complex formation (starred phosphates).
ferent interference effects on each strand, again providing valuable informa-
tion on the structure of the protein–DNA complex.
The two nonesterified oxygens at each phosphate are nonequivalent diaste-
reoisomers, one of which projects toward the major groove while the other
Ethylation Interference 239
projects toward the minor groove. Although, upon modification, both isomers
will show the same charge-neutralization effects, the steric effects on protein–
nucleic acid complex formation will be markedly different. The effect has been
investigated by incorporation of unique diastereoisomers of ethyl and methyl
phosphonates into synthetic oligonucleotides. These modified target sites have
then been used in binding assays for restriction enzymes and repressors using
standard gel retardation or filter binding assays. Ethylated phosphotriesters
have been shown to reduce the T
m
of a d[GGAA(Et)TTCC] duplex with the
Rp–Rp duplex melting 11ºC lower than the unmodified duplex, as a result of
steric clashes (16). Therefore, incorporation of methyl phosphonates may be a
better approach and has been used to probe lac repressor–operator interactions.
Modification of phosphates outside the binding site had no effect on complex for-
mation, whereas sites shown to be important by biochemical assays showed differ-
ent effects for each isomer. In one case, the alkyl group blocked direct interaction
with the phosphate or close approach of the protein, whereas in the other case,
ionic or hydrogen bonded interactions with phosphate remained possible (17).
4. Notes
1. Modification at secondary (nonphosphate) sites: Early work on the reaction of
alkylating agents with DNA showed that a number of products are obtained. With
EtNU, phosphotriester groups comprise 60–65% of the reaction products (9).
The remaining products are the result of reactions at base oxygen groups with
relative abundance in the order thymine O2 = guanine O6 > thymine O4 >>
cytosine O2 for double-stranded DNA (12). To our knowledge, the effects of
such modifications on DNA binding by proteins have not been addressed in the
literature on ethylation interference experiments. However, some prediction of
the effects might be made based on a knowledge of the structure of DNA. Thym-
ine O2 and cytosine O2 are in the minor groove, and guanine O6 and thymine O4
are in the major groove. For sequence-specific DNA-binding proteins interacting
with DNA via the major groove, it would be expected that the presence of an
ethyl group would inhibit complex formation, but that a modification in the minor
groove would be less inhibitory.
2. Level of modification of DNA by EtNU: A similar intensity of each band (sub-
ject to the position-dependent variation of reactivity observed with EtNU) is the
required level of modification. We have used a single batch of “home-produced”
EtNU for all our ethylation interference experiments. The conditions of time and
temperature used with this batch give an appropriate level of probe modification.
There may be variations in the reactivity of EtNU from different sources leading
to undermodification or overmodification. The correct modification conditions
can readily be determined by performing a test ethylation on a small amount of
DNA, such as 20,000 cpm, followed by alkaline cleavage (omitting the gel frac-
tionation step), sequencing gel electrophoresis, and autoradiography. Over-
modification will produce a bias toward short fragments.
240 Manfield and Stockley
3. Choice of fractionation method: The original report of the use of the phosphate
ethylation reaction used the filter binding assay to fractionate protein-bound and
protein-free DNA (2); see also Chapter 1. Filter-bound DNA is then eluted by
washing the filter in a high-salt buffer containing SDS. For other experiments,
we find that TE + 0.1 % (w/v) SDS efficiently elutes DNA from nitrocellulose
filters. The rapid recovery of DNA from filters is an advantage of using the filter
binding assay compared with the gel retardation assay. Despite this, fractionation
by gel retardation assay has proven to be by far the most popular method in the
literature. The advantage of the gel retardation assay is that both the presence and
amounts of multiple complexes can be determined, and these complexes recov-
ered separately, something not possible by filter binding. Parallel binding reac-
tions with ethylated and unethylated DNA and separation on nondenaturing
polyacrylamide gels readily demonstrate any differences in the mobility of com-
plexes formed with each DNA sample.
4. Cofactor requirements: High-affinity DNA binding in our system (MetJ) is
dependent on the presence of S-adenosyl methionine at millimolar concentra-
tions. This cofactor is present in binding reactions and is included in the gel mix,
but it would be prohibitively expensive also to include it in the electrophoresis
buffer. This does not seem to affect the results obtained by this technique. Elec-
trophoresis for extended times does deplete the lower region of the gel of core-
pressor, leading to some complex dissociation. Another feature specific to this
system is the hydrolysis of the corepressor to presumably inactive products. For
this reason, gels were not left to polymerize for more than 2 h.
In other systems where a cofactor is not required or in which the cofactor is
cheap and/or stable over extended periods, the conditions used for the gel-
retardation-assay fractionation step should be optimized by a consideration of
the specific features of the system under study. For further details, consult
Chapter 2.
5. Effects of salt precipitates: During ethanol precipitation of DNA, salt is often
also precipitated. This white crystalline precipitate is readily distinguished from
the almost clear nucleic acid pellets. The interaction of DNA-binding proteins
with their sites is strongly ionic strength dependent, and therefore the presence of
a high concentration of salt following ethanol precipitations will inhibit complex
formation in addition to any ethylation interference effects. After the cleavage
reaction at modified sites, another ethanol precipitation is performed. The pres-
ence of a large amount of salt at this stage will prevent complete dissolution of
the pellet and will interfere with subsequent electrophoresis. A dark background
between each band was often observed on autoradiographs. It is believed that this
is caused by the presence of salt in the sample. Reprecipitation as described below
helps to reduce this problem.
To remove any salt precipitate, the pellet can be dissolved in a small volume
of TE (e.g., 100 µL) and precipitated by addition of 2 volumes of ethanol without
addition of further amounts of salt. The DNA can be pelleted as described in step
4 in Subheading 3.2.3.
Ethylation Interference 241
6. Recommended controls for the gel-retardation-assay (EMSA) fractionation step:
With a precomplex formation modification reaction such as ethylation, it is
important to perform appropriate control reactions especially for the gel retarda-
tion step. For such binding reactions, we use 20,000-cpm unmodified DNA in the
presence and absence of MetJ at the concentration used in the binding reaction
with ethylated DNA samples. The specific activity of protein samples may vary
from batch to batch. The control reaction outlined above will confirm that the
protein sample used is active for DNA binding. In gel retardation assays, the
mobility of complexes is a function of a number of properties of the system, such
as charge and molecular weight of the protein, stoichiometry of the complex, and
bending of the DNA induced by binding of the protein. Demonstration that the
mobility of complexes formed with ethylated and unethylated DNA is the same
is probably good evidence that there are no significant differences between
the complexes.
The protein concentration used in the fractionation step dictates how many
interfering sites are reported. Because the ethyl groups at different sites affect
protein binding to differing degrees, increasing the protein concentration can
mask any weak interference effects such that only the most strongly interfering
sites will be detected. Comparison of the yield of complex on modified DNA
with the control complex on unmodified DNA will show the level of binding-site
saturation and, therefore, indicate the level of discrimination between strongly
and weakly interfering sites that can be expected. Using a range of protein con-
centrations in the binding reaction with aliquots of the ethylated DNA should
therefore allow the strength of the inhibitory effect at each site to be placed in
rank order. This is valuable structural information, as it might be expected that
the strongest effects will be observed at sites that are in closest contact to the
protein in the complex.
7. The presence of multiple cleavage products at each phosphate: The products
of the cleavage reaction at phosphotriester groups carry either 3'-OH or
3'-ethylphosphate groups. For large fragments on low-percentage polyacrylamide
gels, these two species are not resolved. However, for short fragments on high-
percentage gels, two bands are observed at each residue. In practice, this does not
produce problems with data analysis.
8. Troubleshooting: We have experienced few problems with this technique. Most
problems have been associated with the specific properties of the proteins we
have studied. However, it is possible to envisage a number of potential problems
and explanations for these, and remedies are presented here.
Of the available structures of DNA-binding proteins complexed to DNA frag-
ments, there are none in which the protein does not make some contacts to the
phosphodiester backbone. Thus, it is expected that because of the size of the
ethyl group, an interference effect will always be observed. In the event that no
inhibition of complex formation is observed, it should be confirmed that
ethylation has occurred by performing a titration of the ethylation reaction as
discussed in Note 2.
242 Manfield and Stockley
It is possible that an increase in free DNA is observed on the gel retardation
(EMSA) assay but that no cleavage products are observed on the sequencing gel,
although the full-length DNA is present. This could be caused by an error with
the buffer used to resuspend the eluted DNA pellet, the NaOH solution used to
perform strand scission, or the temperature of the reaction, all of which can be
readily checked.
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