Chapter 6

The Use of Diethyl Pyrocarbonate and Potassium Permanganate as Probes for Strand Separation and Structural Distortions in DNA
Brenda F. Kahl and Marvin R. Paule
Summary
Diethyl pyrocarbonate (DEPC) and potassium permanganate are useful reagents for detecting DNA distortions, especially melted regions. Unlike most other footprinting methods, these reagents can detect such distortions even within the regions of proteinDNA complexes normally protected in other footprinting techniques. Further, reactions are very robust, so that distorted regions can be detected even under conditions where efficiency of DNAprotein complex formation is not high. DEPC reacts with bases that are fully or partially unstacked in DNA, in the preferential order adenosine > guanine >> cytosine. Permanganate reacts strongly with thymine in unstacked regions of DNA, and exhibits only very weak reaction with guanine, cytosine, or adenine. The combination of both reagents gives excellent coverage of all sequence regions of DNA. Because reaction requires unstacking, the two reagents detect both melted regions and regions that are unstacked because of other distortions such as bending. Permanganate has the additional advantage that it can be utilized in living cells. Key words: DNA, Footprinting, Melted DNA, DNA bending, Dithylpyrocarbonate, Permanganate.

1. Introduction
In the search for methods to explore the interaction between proteins and DNA, a plethora of footprinting techniques have been developed and many of which are discussed elsewhere in the present work. Most footprinting techniques are based on the simple premise of specific DNA regions being protected from the reagent by the bound protein or molecule of interest. However,

Tom Moss and Benot Leblanc (eds.), Methods in Molecular Biology, DNA-Protein Interactions, vol. 543 Humana Press, a part of Springer Science + Business Media, LLC 2009 DOI: 10.1007/978-1-60327-015-1_6

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a number of studies over the past decade have revealed remarkable distortion of the DNA molecule, including bending and strand separation, in response to the bound protein. These distortions are often within the classical footprint, but are rarely detected by these classical techniques. Thus, their detection requires alternative approaches. Unlike enzymatic methods, the chemical probes diethyl pyrocarbonate (DEPC) and potassium permanganate can access and react with the entire sequence of the DNA, distinguishing DNA distortions under the foot of a typical footprinting experiment. Combining the information obtained from these chemical probing techniques with the spatial information afforded by traditional footprinting gives an in-depth account of the various ways proteins and other molecules interact with DNA. DEPC and potassium permanganate are useful probes because of their preferential reactivity with single-stranded over double-stranded DNA. In addition, unlike typical footprinting techniques where near saturation of the DNA with protein is necessary to observe a clean footprint, potassium permanganate and DEPC probing does not require most of the DNA to be in complex with the protein or molecule of interest. Due to the sensitivity of these chemical probes, it is possible to observe reactive bases even when the population of single-stranded DNA is very small. The mechanism by which DEPC modifies bases has been investigated, but is still not clearly understood (13). DEPC predominantly reacts with purine residues, but may react weakly with cytosine residues as well. DEPC modifies DNA by an outof-plane attack on several of the nucleophilic centers in purines, leading to the scission of the glycosidic bond. Double stranded B-form DNA does not undergo modification by DEPC because the close stacking of neighboring bases occludes access to the out-of-plane surfaces. However, under conditions where the conformation deviates from B-form (such as strand separation or bending), purines in the sequence become more accessible to modification by DEPC. Carbethoxylation of the imidizole ring N-7 produces strand scission under alkaline conditions. Thus, DEPC is commonly used to detect purines which are present in melted or distorted DNA sequences. While DEPC can react with both purines, it shows a marked preference for adenines over guanines in most instances. Potassium permanganate reacts with double bonds, oxidizing them to vicinal diols. In nucleic acids, the base thymine is oxidized most vigorously, while reaction with C, G, and A is minimal. The mechanism behind this preferential reactivity is believed to arise from an out-of-plane attack on the 5,6-double bond of the thymine ring (49). Although the ring is still intact, the loss of aromaticity resulting from insertion of hydroxyl groups on the 5- and 6- carbons leads to a reduction in hypochromicity.

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Treatment of the vicinal diol with strong base leads to ring opening and cleavage of the phosphodiester backbone. As with DEPC, stereochemical hindrance from base stacking prohibits reactivity of double stranded B-form DNA. In DNA, which is denatured or is altered from B-form, the thymine ring becomes susceptible to modification by potassium permanganate. Because of their base preferences, the combined use of DEPC and potassium permanganate allows complete analysis of both GC- and AT-containing sequences in DNA. DEPC and potassium permanganate modification have been used to detect a number of distorted DNA structures in both prokaryotic and eukaryotic cells, including open complex formation during transcription (1014), steps in promoter clearance (1517), elongation (1719), and termination (20, 21), RNA DNA hybrid structures in transcription elongation complexes (22, 23), drug binding to DNA (2426), chromatin positioning (27, 28), recombination events (2931), and single-stranded binding protein binding domains (3234). On DNA alone, these reagents can reveal sequence-dependent distortions (3537), negatively supercoiled DNA (3840), cruciform DNA structures (41, 42), DNA hairpins such as those found in triplet expansion diseases like fragile X-syndrome (43, 44), and Z-DNA, H-DNA, or triplex DNA (4548). Figures 1 and 2 give examples of the

Fig. 1. Potassium permanganate sensitive sites when an open promoter complex is formed on the coding strand by RNA polymerase I from Acanthamoeba castellanii. Lane M contains a G + A MaxamGilbert sequencing ladder, lane 1 contains DNA exposed to KMnO4 treatment in the absence of any proteins, and lane 3 contains DNA with the addition of proteins necessary for melting of DNA. Numerical designations refer to the transcription start site. Hypersensitive sites in bold denote regions of strand separation.

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Fig. 2. Potassium permanganate and DEPC probing of stalled transcription complex. (a) Potassium permanganate probing of the coding strand when RNA polymerase I is paused at +31 from the transcription start site. Lane M contains G + A MaxamGilbert sequencing ladder, lane 1 contains DNA alone with KMnO4 treatment, and lane 2 displays the permanganate sensitive sites when RNA polymerase from Acanthamoeba castellanii is paused at +31 from the transcription start site. The hypersensitive sites in bold at positions +21, +22 and +32, +33 define the transcription bubble on the coding strand. Hyposensitive thymidines in the transcription bubble depict protection due to the formation of an RNADNA hybrid. (b) DEPC probing on the noncoding strand when RNA polymerase I is paused at +31 from the transcription start site. Lanes are the same as in (a). DEPC sensitive sites in bold define the leading edge of the transcription bubble on the noncoding strand. Unreactive As in the region may be due to protein interference or the slightly less reactive nature of DEPC compared to KMnO4.

data which can be obtained. Figure 1 shows one of the more common uses of potassium permanganate, the analysis of open promoter complex formation. Figure 2 shows results for both DEPC and potassium permanganate acting on a stalled transcription elongation complex, showing both the melted transcription bubble and the unreactive RNADNA hybrid. Virtually any sequence that deviates from B-form DNA is a candidate for probing with DEPC and potassium permanganate. Since DEPC and potassium permanganate only modify the susceptible bases without cleaving the phosphodiester backbone, further steps need to be taken to visualize the positions of the modified bases. There are three different methods commonly used: The first, which can only be used for experiments performed in vitro, utilizes 5 or 3 end-labeled DNA fragments. After treatment with DEPC or potassium permanganate, the DNA is treated with piperidine to cleave the phosphate backbone on the 3 side of the modified nucleotide. This procedure is useful when comparing the results from multiple footprinting

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techniques, since the same fragment of labeled DNA may be utilized with each type of experiment. This method also works well when examining short tracts of DNA or DNA which does not amplify well in a thermocycler. The other two methods, primer extension and thermocycle amplification, are used when working in vivo (potassium permanganate only) or with circular pieces of DNA in vitro. Thermocycle amplification is particularly beneficial when working with limited amounts of DNA (in the low nanogram range) or when the ratio of proteinDNA complex to DNA is low. Subheadings 2 and 3 are general guidelines for probing with DEPC and potassium permanganate. 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 conditions for each particular application, and guidelines for this can be found in Subheading 4.

2. Materials
1. Potassium permanganate, DEPC, piperidine, and 2-mercaptoethanol can be purchased from Sigma-Aldrich. DEPC, piperidine, and 2-mercaptoethanol should be stored at 4C 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 20C until ready for use: 1 M HEPES pH 7.9. 1 M MgCl2. 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-methylenebisacrylamide). 10 mg/mL Proteinase K. 10% SDS. DEPC Stop Buffer: 0.2% SDS, 0.6 M sodium acetate pH 5.2.

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TE: 10 mM TrisHCl pH 8.0, 1 mM EDTA. Electrophoresis loading buffer: 0.1% bromophenol blue, 0.1% xylene cyanol, 10 mM EDTA, 80% deionized formamide. 3. A 300 mM stock solution of KMnO4 can be made by heating 2.73 g of KMnO4 in 50 mL of deionized water. The solution can be stored at room temperature in a brown bottle for 1 month. 4. 10 reaction buffer, for example: 200 mM HEPES pH 7.9, 100 mM MgCl2, 1 mM DTT, 1 mg/mL BSA, 100 mM KCl. The choice of this buffer will depend upon the conditions needed to form the complex under study. 5. Protein dilution buffer, for example: 20 mM HEPES pH 7.9, 1 mM EDTA, 10% glycerol. Choice will dependent 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. 10 neutralization buffer: 0.5 M HEPES pH 7.9, 0.1 M MgSO4, 2 mM DTT. 10. dNTP mix: 5 mM of each dNTP.

3. Methods
Experiments using DEPC or potassium permanganate follow similar protocols. The only exceptions are the addition of 2-mercaptoethanol to quench reactions using potassium permanganate, the duration of the DNA modification 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 precurser either by agarose gel electrophoresis or by size exclusion column chromatography, and stored in TE. 2. In a 1.5-mL siliconized microcentrifuge tube, add 4 mL 10 reaction buffer, 40,000 cpm of DNA, and sterile deionized water to a volume of 20 mL. Proteins, diluted in an appropriate buffer, are added to the reaction to give a final volume of 40 mL (see Note 1). Incubate for the desired amount of time to form DNAprotein complexes. 3. Modifying the susceptible bases (see Notes 24):

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KMnO4treatment. Add freshly diluted potassium permanganate to give the appropriate concentration and incubate for desired time period. For example, adding 2 mL of 100 mM KMnO4 (approximately 9 mM final concentration) for 2 min seems to work well for detecting melted DNA in transcription initiation complexes. DEPC treatment. Add 1 mL 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: KMnO4treatment. Quench the reaction by adding 3 mL of 2-mercaptoethanol, vortex and place on ice. Add 45 mL of 0.2% SDS, 2 mg/mL proteinase K and incubate at 50C for 1 h. Add 90 mL of 0.6 M sodium acetate, 300 mg/mL linear polyacrylamide and 2.5 volumes 95% ethanol. Mix and centrifuge for 30 min at 14,000 rpm (16,000g) in a microfuge. Remove supernatant and wash with 150 mL of 70% ethanol. Centrifuge 5 min as above. Remove supernatant and dry the pellet on medium heat for 5 min in a Speed Vac. DEPC treatment. Stop the reaction by adding an equal volume of DEPC Stop Buffer and phenolCHCl3 extract. Add 5 mL 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 the pellet on medium heat for 5 min in a Speed Vac. 5. Alkaline cleavage: Suspend the pellet in 50 mL of 1 M piperidine (10% v/v) and incubate at 90C for 30 min. Place a lead weight on top of the tubes or use tube locks to prevent the lids from opening. 6. Place tubes on ice to cool and centrifuge briefly. Add 50 mL of 0.6 M sodium acetate, 300 mg/mL linearized polyacrylamide, and 250 mL of 95% ethanol. Mix and centrifuge for 30 min as above. Wash the pellet with 150 mL of 70% ethanol. Spin samples for 5 min. Remove supernatant. 7. To remove residual piperidine, add 30 mL of sterile deionized water to each sample and dry on medium heat in a Speed Vac (see Note 5). 8. Add 5 mL of electrophoresis loading buffer, vortex samples for 30 s and heat samples at 95C for 3 min. Place samples on ice. 9. Load samples on sequencing gel and analyze by standard methods (see Notes 6 and 7).

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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 PCR. DEPC cannot be used for in vivo experiments because of its low solubility. The procedure for in vivo modification is fairly straightforward, however, 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 treatment 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 KMnO4 (typically in the low mM range, depending upon the medium, approximately 1020 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 the cells in a cold Sorvall SS34 rotor for 5 min at 5,000 rpm (3,000g). 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 (usually 220 mM) for the necessary period of time (10 s to 5 min). Stop the permanganate 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 (4951). Purified modified DNA should be adjusted to a final concentration of approximately 15 ng/mL and be free of contaminants that interfere with extension reactions. Extractions involving phenol should be repeated 34 times or until there is no contaminating interphase. Modifications to the DNA can then be visualized by PCR amplification detailed in Subheading 3.4. An alternative method for identifying modified genomic DNA is the use of ligation mediated PCR (LMPCR) (52, 53).

3.3. Primer Extension Analysis of DNA Treated In Vitro or In Vivo

Since DNA modified in vivo or when in circular form is not endlabeled, 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 24 of Subheading 3.1, substituting

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20500 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 (20500 ng for in vitro and 500 ng for in vivo studies), add 0.30.5 106 cpm of 5 endlabeled primer and dilute to 36 mL with distilled water. 2. Add 4 mL of 0.01 M NaOH to each reaction and mix well. 3. Denature DNA by heating to 95C for 2 min. 4. Add 5 mL of 10 neutralization buffer and mix. 5. Hybridize primer to DNA by heating sample for 3 min at or just under the calculated Tm of the primer. 6. Add 5 mL of a solution containing all four dNTPs at a concentration of 5 mM each. 7. Add 0.51.0 unit of the Klenow fragment of DNA polymerase I and mix gently. Incubate tube for exactly 10 min at 50C. 8. Quench by adding an equal volume (~50 mL) of 0.6 M sodium acetate, 20 mM EDTA, and place on ice. 9. Precipitate DNA by adding 300 mL of 95% ethanol, mix and centrifuge for 30 min. Wash the pellet with 150 mL of 70% ethanol. Centrifuge for 5 min, remove supernatant, and dry the pellet. 10. Suspend the pellet in 5 mL of 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 mL 10 reaction buffer supplied with the thermostable polymerase 2 mL of dNTP mix 0.5 106 cpm end-labeled primer Distilled water to a final volume of 49.5 mL. 2. Program thermocycler. For example: 1 Round: 2 min at 95C, pause and add 0.5 mL of thermostable polymerase (2.5 units/ml) 30 s at Tm of primer 30 s at 72C 1520 Rounds: 1 min at 95C 30 s at Tm of primer 30 s at 72C 1 Round: 5 min at 72C.

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3. Precipitate DNA by adding 50 mL of 0.6 M sodium acetate, 300 mg/mL of linear polyacrylamide, and 250 mL of 95% ethanol. Mix and centrifuge for 30 min at 14,000 rpm (16,000g) in a microfuge. Wash the pellet with 150 mL of 70% ethanol, centrifuge for 5 min. Remove the supernatant and dry the pellet on medium heat for 5 min in a Speed Vac. 4. Suspend the pellet in 5 mL of 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 procedure 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 varying amounts of time may be necessary. Too little potassium permanganate results in no signal, and too much potassium permanganate can result in a high background. 25 mM 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 permanganate 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 mL of deionized water and drying as described in step 7 of Subheading 3.1.

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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 adjacent 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 due to (a) improper primer sequence; (b) annealing temperature higher than Tm ; (c) contaminants present in reaction; and (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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