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Chapter 31

The Cruciform DNA Mobility Shift Assay: A Tool to Study Proteins that Recognize Bent DNA
Victor Y. Stefanovsky and Tom Moss
Summary
So-called architectural DNA binding proteins such as those of the HMGB-box family induce DNA bending and kinking. However, these proteins often display only a weak sequence preference, making the analysis of their DNA binding characteristics difficult if not impossible in a standard electrophoretic mobility assay (EMSA). In contrast, such proteins often bind prebent DNAs with high affinity and specificity. A synthetic cruciform DNA structure will often provide an ideal binding site for such proteins, allowing their affinities for both bent and linear DNAs to be directly and simply determined by a modified form of EMSA. Key words: Architectural proteins, HMGB-box protein, Bent DNA, Cruciform, EMSA.

1. Introduction
The first reported interaction of an HMGB-box protein (HMGB1) with a bent stable synthetic DNA structure suggested that this family of proteins displayed an intrinsic affinity for bent DNA (1). The assay was based on the electrophoretic mobility shift (EMSA) of an in vitro assembled synthetic cruciform when it was bound by an HMGB box. It was found that binding to the prebent cruciform DNA occurred with a much higher affinity and specificity than to linear DNA. Shortly after, the same group demonstrated an identical behavior for the sequence-specific transcription factor SRY, which contains a single HMG box (2), followed by similar reports on the other HMGB-box proteins LEF-1/TCF 1 (3, 4). The RNA Polymerase I transcription
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_31

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factor UBF, like HMGB1, is a nonsequence-specific DNA binding protein with multiple HMGB boxes, and as such its binding properties were difficult to analyze by EMSA using linear DNA. However, it was found to bind strongly to cruciform DNA (5). A detailed study showed that the individual HMGB boxes 1 and 2 of UBF, responsible for the rDNA promoter in-phase bending and enhancesome formation (6, 7), each bind with high affinity to cruciform structures (8). Moreover, competition with linear DNA proved to be a valuable tool for detecting changes in the binding affinity of these boxes as a result of mutations or posttranslational modifications. Such changes are believed to reflect structural changes in the proteinDNA complex due to an altered bending capacity of the HMGB boxes. The 14-3-3 family of proteins were found to specifically recognize cruciform structures at origins of DNA replication in a cell-cycle dependent manner and were identified as regulators of DNA replication (9), and the cruciform mobility shift assay has also been used for analyzing these proteins. The cruciform mobility shift assay has, thus, shown itself to be a useful tool in the study of proteins that display enhanced affinity for bent DNA. Here we present the basic assay using the HMGB boxes as example.

2. Materials
1. Stock solutions of cruciform oligonucleotides at 10 pmoles/ml in ddH 2O;Oligo 1 5 -CCCTATAACCCCTGCATTGAAT TCCAGTCTGATAA-3 Oligo 2 5-GTAGTCGTGATAG GTGCAGGGGTTATAGGG-3 Oligo 3 5-AACAGTAGCT CTTATTCGAGCTCGCGCCCTATCACGACTA-3 Oligo 4 5-TTTATCAGACTGGAATTCAAGCGCGAGCTCGAATAAG AGCTACTGT-3 These oligonucleotides are designed in order to anneal with each other and form a cruciform structure, Fig. 1. 2. [g-32P]-ATP (Perkin-Elmer). 3. T4 Polynucleotide Kinase (NEB). 4. 7.5 M ammonium acetate. 5. 95 and 70% ethanol. 6. TMS annealing buffer: 100 mM NaCl, 10 mM TrisHCl of pH 7.5, 10 mM MgCl2. 7. 2 Binding buffer: 16% Ficoll, 200 mM NaCl, 20 mM HEPES of pH 7.9, 10 mM KCl, 2 mM EDTA, 2 mM spermidine, 1 mM DTT. 8. TBS:10 mM TrisHCl of pH 7.5, 100 mM NaCl.

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Fig. 1. Schematic formation of a cruciform structure. The arrows indicate the 3-termini of the annealed oligonucleotides 1, 2, 3, and 4; The asterisk indicates the radioactive 32 PO4 group on oligonucleotide 3.

9. TBE (10) Prepare 1 L by mixing 108 g Tris-base, 55 g boric acid, 40 ml 0.5 M EDTA, pH 8.0. 10. 40% acrylamide stock solution (38 parts acrylamide: 2 parts bisacrylamide). 11. 0.1% Xylene cyanol. 12. Gel-loading buffer (10) 0.25% bromophenol blue, 0.25% xylene cyanol, 25% Ficoll 400. 13. Prepare multiple 0.5-ml Eppendorf tubes with pierced bottom (make several holes with an 1821-gauge syringe needle) and fill the bottom of the tubes with glass wool (keep stock in 70% ethanol). Autoclave and keep in a sterile jar until use. 14. For competition experiments: double-stranded linear DNA containing the target DNA sequence of interest, stock solution at least 200500 mg/ml in ddH2O. 15. For antibody supershift: specific antibodies against the protein of interest in serum dilutions ranging from undiluted to 1:1,000 in TBS.

3. Methods
3.1. 5 End Labeling of Oligonucleotide 3

1. Take 2 ml (20 pmoles) oligo 3 and add 2 ml 10 Polynucleotide Kinase buffer (NEB) 10 ml [g-32P]-ATP, 5 ml H2O and 1 ml T4 Polynucleotide Kinase (~10 units) (NEB) 2. Incubate for 1 h at 37C.

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3. Add 10 ml 7.5 M ammonium acetate (final concentration: 2.5 M). 4. Add 270 ml 95% Ethanol. 5. Leave 5 min, at RT. 6. Spin in an Eppendorf centrifuge 5 min at 13,000 rpm. 7. Carefully discard supernatant into radioactive waste, add 300 ml 70% ethanol. 8. Spin 1 min 13,000 rpm. 9. Carefully discard supernatant into radioactive waste.
3.2. Annealing of the Oligonucleotides and Isolation of the Labeled Cruciform

1. Add 2 ml (20 pmoles) each of oligos 1, 2, and 4 to precipitated, labeled oligo 3. 2. Add 25 ml TMS and mix thoroughly to redissolve oligo 3. 3. Take a small aliquot to determine approximate specific activity, that is total Cerenkov cpm/20 pmole oligo 3. 4. Place in an aluminum heating block at 90C, switch off heating immediately, and insulate by covering with a Styrofoam box. Leave for 3 h to anneal cruciform. Alternative method: Leave 3 min at 90C, then 10 min at 68C, and 30 min at 37C. This can be easily performed in a thermal cycler. 5. Add 3 ml 10 gel-loading buffer. 6. Load into a 1-cm wide pocket on a 1-mm-thick, 20-cm-long 6.5% polyacrylamide gel in 1 TBE. 7. Run at 10 V/cm for 23 h until bromophenol blue has migrated about 14 cm. 8. Remove the upper plate of the gel, cover with Saran wrap, and autoradiograph for 30 s to 1 min. Ideally, use fluorescent ink markers to allow realignment of film to gel. Alternatively, use one corner of a radiography cassette to align the film with the gel plate during exposure. 9. Realign the developed film under the gel on a transilluminator and excise the cruciform band using a scalpel. The cruciform migrates slightly below the xylene cyanol band on a 6.5% gel (see Fig. 2 and Note 1).

3.3. Extraction of Cruciform

1. Place the cut gel fragment containing the cruciform in a 0.5ml Eppendorf tube. Pierce several holes through the bottom of the tube with a gauge 1821 syringe needle. Place the tube in a 1.5-ml Eppendorf tube without lid. 2. Centrifuge 2030 s or until the gel passes through the holes into the bottom tube forming a gel pellet.

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Fig. 2. Migration of the cruciform on the preparative gel. The cruciform structure was resolved on a 6.5% polyacrylamide gel and exposed as described in Methods. Left panel: position of the cruciform (X-form) relative to the dyes (XC xylene cyanol and BPB bromophenol blue) indicated by black ellipses. Right panel: an example of incomplete annealing. Positions of the monomer and dimer forms are indicated on the left side of the figure.

3. Add 300 ml TMS to the pellet to form a slurry. 4. Seal the tube and leave overnight at 4C. 5. Put a previously prepared glass wool-bottomed, pierced, autoclaved 0.5-ml tube (see Subheading 2, item 13) in an intact 1.5-ml Eppendorf without lid. 6. To recover eluate, transfer all the gel slurry into the 0.5-ml tube using a 1-ml pipette with large tip opening (cut-off) and centrifuge for 20 s. 7. Adjust the eluate volume to 400 ml with TMS. The eluted cruciform DNA should be at ~50 fmole/ml and nearly all the radioactivity should have been eluted. Cerenkov count a 2-ml aliquot in order to calculate the concentration of cruciform using the specific activity calculated from Subheading 3.2, step 3. Stored at 4C the labeled cruciform is stable for at least a week. You will need about 100 fmoles (2 ml) per mobility shift assay.

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3.4. Cruciform Mobility Shift Assay

Each mobility shift reaction is performed in 10 ml consisting of 5 ml of 2 Binding Buffer, 2 ml (100 fmole) of cruciform DNA in TMS, and 3 ml of TBS containing varying amounts of the proteins to be assayed. 1. Prepare a 6.5% polyacrylamide gel (acrylamide:bisacrylamide 38:2) at least 1520 cm long and 1-mm thick in 0.5 TBE and use a 0.5% TBE as running buffer. Prerun the gel for 1 h at 11 V/cm. 2. Prepare a stock mix of 2 Binding Buffer (5 ml number of reactions plus one) and cruciform (2 ml (100 fmole) number of reactions plus one). 3. Prepare dilutions of the protein to be analyzed in Eppendorf tubes. Complete the volume of the protein in each tube to 3 ml with TBS. Place 3 ml TBS in a tube to serve as a negative control. Since the Kd of the expected complexes is typically less than mmolar, start with protein amounts ranging from 2 to 30 pmoles. 4. Add 7 ml binding buffer/cruciform mix, as in step 2, to each protein dilution. 5. Incubate 1030 min at room temperature. 6. Add 0.5 ml 0.1% xylene cyanol to each sample and load onto the gel. 7. Electrophorese for 34 h at 11 V/cm. 8. Transfer the gel onto a sheet of Whatman 3-MM paper and cover with Saran wrap. 9. Dry the gel for 30 min at 85C. 10. Expose the gel to radiography film or use a commercial phosphoimaging device to detect cruciform and analyze the results. A typical example of the electrophoretic separation is shown in Fig. 3 and Notes 2 and 3.

3.5. Competition with Linear DNA

The affinity of the protein for linear relative to cruciform DNA can be determined in a simple competition assay (see Fig. 4). Typically, a 1- to 10-fold molar excess of the linear DNA fragment over the protein is required. Add 1 ml of the double-stranded linear fragment to the cruciform DNA before mixing with the protein sample in step 3 of Subheading 3.4, then proceed as described in that section. A protein concentration that yields less than complete shifting of the cruciform in the absence of linear DNA must be used if the relative affinity of the protein for linear DNA is to be determined. In the case of impure protein samples, it may be necessary to determine the identity of the protein that is responsible for the cruciform shift. This can sometimes be achieved by up-shifting

3.6. Supershifting with a Specific Protein Antibody

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Fig. 3. A typical example of cruciform shift assay. About 100 fmoles of cruciform was incubated with the indicated amounts of HMGB box1 from UBF, see Subheading 2. The proteinDNA complex (complex) and the naked cruciform (X-form) are indicated.

Fig. 4. Competition with linear target sequence. The cruciform structure was incubated with 20 pmoles of UBF HMGB box1(as in Fig. 3) and with increasing amounts of linear human rDNA promoter UCE fragment, see ref. 8. In this case the cruciform shift is efficiently competed with 200 pmoles of the linear promoter fragment. Complex and X-form as in Fig. 3.

the cruciform-protein complex by the addition of an antibody before electrophoretic analysis. Add 1 ml of an appropriate range of antibody dilutions to the samples just before applying them to the gel, then proceed as described in that section. An example of such an upshift assay is shown in Fig. 5.

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Fig. 5. Specific antibody supershift. The cruciform structure (100 fmoles) was incubated with 20 pmoles of UBF HMGB box1 and 1 ml diluted polyclonal anti-UBF antibody as indicated. An increasing large supershift is observed with increase in antibody concentration, eventually producing antibody-proteinDNA complexes high in the gel indicated by arrows.

4. Notes
1. Annealing of the cruciform is not complete (see Fig. 1B, right panel). Usually, the annealing is so efficient that only the completed cruciform, containing all four oligos, is visible on the gel. In some cases, however, smaller structures containing one or two oligos may be present, running closer to the bromphenol blue. If the cruciform is not the major product:i) Repeat the annealing after verifying the identity and quality of oligonucleotides. ii) Check the concentration of your oligonucleotides. Equimolar amounts must be used. iii) The gel may have been run too hot. Check the voltage, it should not exceed 11V/cm. 2. More than one shifted band is visible on the gel. There may be a second cruciform binding protein in the protein preparation. A cruciform in some cases may bind more than one protein molecule. See also Note 3. 3. Certain protein-DNA complexes migrate faster than the naked cruciform (downshift). Dependent on buffer conditions cruciforms may adopt alternative tertiary folds, affecting

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Fig. 6. Differential migration of the proteincruciform DNA complex depending on buffer conditions. Increasing amounts of UBF HMGB box2 were incubated with the cruciform using two different binding buffers: buffer A, the standard binding reaction (2 mM MgCl2 final concentration) and buffer B, as buffer A but giving final concentrations of 5 mM MgCl2 and 0.3 mM ATP. Positions of the free cruciform (X-form), the upshift and the downshift are indicated with arrows.

their mobility during electrophoresis. For example, HMGBbox 2 of UBF under standard buffer conditions gave a downshift as well as an upshift. However, addition of an extra 3 mM MgCl 2 during the binding reaction eliminated the downshift, see Fig. 6. It is possible that acrylamide concentration or bisacrylamide:acrylamide ratio in the gel may also affect relative migration of cruciform structural isomers.

Acknowledgments
This work was supported by an operating grant from the Canadian Institutes of Health Research.

References
1. Bianchi, M. E., Beltrame, M., and Paonessa, G. (1989). Specific recognition of cruciform DNA by nuclear protein HMG1, Science 243, 10561059. 2. Ferrari, S., Harley, V. R., Pontiggia, A., Goodfellow, P. N., Lovell-Badge, R., and Bianchi, M. E. (1992). SRY, like HMG1, recognizes sharp angles in DNA, EMBO J. 11, 44974506. 3. Lilley, D. M. (1992). DNAprotein interactions. HMG has DNA wrapped up, Nature 357, 282283. 4. Van de Wetering, M., and Clevers, H. (1992). Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix, EMBO J. 11, 30393044. 5. Kuhn, A., Stefanovsky, V., and Grummt, I. (1993). The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription, Nucleic Acids Res. 21, 20572063. 6. Bazett-Jones, D. P., Leblanc, B., Herfort, M., and Moss, T. (1994). Short-range DNA looping

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by the Xenopus HMG-box transcription factor, xUBF, Science 264, 11341137. 7. Stefanovsky, V. Y., Bazett-Jones, D. P., Pelletier, G., and Moss, T. (1996). The DNA supercoiling architecture induced by the transcription factor xUBF requires three of its five HMGboxes, Nucleic Acids Res. 24, 32083215. 8. Stefanovsky, V. Y., Pelletier, G., Bazett-Jones, D. P., and Moss, T. (2006). ERK modulates

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