Probing DNA Structure with Hydroxyl Radicals

Thomas D. Tullius1

1 Boston 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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Table of Contents

  • Basic Protocol 1: 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
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: 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 ) 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 (H 2O 2)
  • 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 protocol 2), and image analysis (optional; see protocol 3)

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

  Materials
  • Gel (see protocol 1)
  • 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 protocol 1)
  • Personal computer running software for image analysis:
  •  ImageQuant (Molecular Dynamics)
  •  Origin, with Peak Fitting Module (OriginLab)
  •  Microsoft Excel (optional)
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Figures

Videos

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 TnAn 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.
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