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Probing DNA Structure with Hydroxyl Radicals

Thomas D. Tullius¹

¹Boston University, Boston, Massachussetts

Publication Name:

Current Protocols in Nucleic Acid Chemistry

Unit Number:

Unit 6.7

DOI:

10.1002/0471142700.nc0607s07

Online Posting Date:

February, 2002

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Abstract

The hydroxyl radical is a useful probe for studying the shape of the surface of a DNA molecule. Using this technique, fine details of DNA structure can potentially be revealed. This unit describes how to use the hydroxyl radical to generate a random cleavage pattern at the surface of the molecule, separate the broken DNA strands by polyacrylamide gel electrophoresis, and analyze the cleavage pattern to give an image of the surface of the molecule.

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Unit Introduction
Basic Protocol: Preparation of the Hydroxyl Radical Cleavage Pattern of a DNA Molecule
Support Protocol 1: Drying a High-Percentage Polyacrylamide Denaturing Gel
Support Protocol 2: Analysis of the Cleavage Pattern
Reagents and Solutions
Commentary
Bibliography
Figures
Tables

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Materials

Basic Protocol: Preparation of the Hydroxyl Radical Cleavage Pattern of a DNA Molecule

Materials

1 ng/µL radiolabeled DNA (5¢-end-labeled with ³²P on one strand; 3000 Ci/mmol; see, e.g., cpmb unit 3.5) dissolved in TE buffer, pH 8.0 (appendix 2A)
1× Fe(II) EDTA (see recipe)
10 mM sodium ascorbate
0.3% (v/v) hydrogen peroxide (H₂O₂)
100 mM thiourea
3 M sodium acetate (appendix 2A)
85% and 100% (v/v) ethanol
Dry ice/acetone bath
Formamide-containing dye mixture (e.g., loading buffer, unit 10.4)
Denaturing poylacrylamide electrophoresis gel (~30 × 40 × 0.04 cm; appendix 3B)
1.5-mL microcentrifuge tubes, siliconized
25° and 90°C heating blocks
SpeedVac evaporator
Phosphor imager with storage phosphor plate that corresponds to the size of the gel
Phosphor imaging plate scanner
Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (PAGE; appendix 3B), drying of high-percentage acryamide denaturing gels (see Support Protocol 1), and image analysis (optional; see Support Protocol 2)

Support Protocol 1: Drying a High-Percentage Polyacrylamide Denaturing Gel

Materials

Gel (see Basic Protocol)
Plastic wrap: 12- or 18-in. (~30- or 45-cm) Saran Wrap or Reynolds film
Whatman 3MM filter paper
Gel dryer

Support Protocol 2: Analysis of the Cleavage Pattern

Materials

Computerized image of gel obtained by scanning the storage phosphor plate (see Basic Protocol)
Personal computer running software for image analysis:
ImageQuant (Molecular Dynamics)
Origin, with Peak Fitting Module (OriginLab)
Microsoft Excel (optional)

Looking for materials? Suppliers Guide

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Figures

Figure 6.7.1

The Fenton reaction of iron(II) EDTA with hydrogen peroxide to generate the hydroxyl radical.

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Figure 6.7.2

Hydroxyl radical cleavage using 5¢- and 3¢-end-labeled DNA. The major product for each is a DNA strand terminated by phosphate at the site of strand breakage. With 3¢-labeled DNA, the aldehyde-terminated strand also is produced in substantial amounts, leading to difficulties in quantitation.

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Literature Cited

Literature Cited
	Balasubramanian, B., Pogozelski, W.K., and Tullius, T.D. 1998. DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc. Natl. Acad. Sci. U.S.A. 95:9738-9743.
	Burkhoff, A.M. and Tullius, T.D. 1987. The unusual conformation adopted by the adenine tracts in kinetoplast DNA. Cell 48:935-943.
	Burkhoff, A.M. and Tullius, T.D. 1988. Structural details of an adenine tract that does not cause DNA to bend. Nature 331:455-457.
	Churchill, M.E.A., Tullius, T.D., Kallenbach, N.R., and Seeman, N.C. 1988. A Holliday recombination intermediate is twofold symmetric. Proc. Natl. Acad. Sci. U.S.A. 85:4653-4656.
	Kimball, A., Guo, Q., Lu, M., Cunningham, R.P., Kallenbach, N.R., Seeman, N.C., and Tullius, T.D. 1990. Construction and analysis of parallel and antiparallel Holliday junctions. J. Biol. Chem. 265:6544-6547.
	Price, M.A. and Tullius, T.D. 1992. Using the hydroxyl radical to probe DNA structure. Methods Enzymol. 212:194-219.
	Price, M.A. and Tullius, T.D. 1993. How the structure of an adenine tract depends on sequence context. A new model for the structure of T_nA_n DNA sequences. Biochemistry 32:127-136.
	Shadle, S.E., Allen, D.F., Guo, H., Pogozelski, W.K., Bashkin, J.S., and Tullius, T.D. 1997. Quantitative analysis of electrophoresis data: Novel curve fitting methodology and its application to the determination of a protein-DNA binding constant. Nucl. Acids Res. 25:850-861.
	Shafer, G.E., Price, M.A., and Tullius, T.D. 1989. Use of the hydroxyl radical and gel electrophoresis to study DNA structure. Electrophoresis 10:397-404.
	Udenfriend, S., Clark, C.T., Axelrod, J., and Brodie, B.B. 1954. Ascorbic acid in aromatic hydroxylation. J. Biol. Chem. 208:731-739.
Key References
	Price and Tullius, 1992. See above.
A comprehensive description of the use of the hydroxyl radical as a probe of DNA structure.
Internet Resources
	http://people.bu.edu/tullius/GelExplorer_Manual.pdf
A detailed online manual that describes the use of the GelExplorer software for quantitative analysis of electrophoresis gel patterns.