Analysis of Oxidized DNA Fragments by Gel Electrophoresis

Mohamad Awada1, Peter C. Dedon1

1 Massachusetts Institute of Technlogy, Cambridge, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.8
DOI:  10.1002/0471142700.nc1008s04
Online Posting Date:  May, 2001
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Abstract

Polyacrylamide gel electrophoresis is used to define and quantify products of deoxyribose oxidation in DNA, based on the unique electrophoretic mobility of DNA fragments possessing deoxyribose oxidation products on their termini. This approach allows initial estimation of the chemistry. Once the chemical identity of damage products has been confirmed, this technique allows sensitive quantitation of the various damage products.

     
 
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Table of Contents

  • Basic Protocol 1: Determination of 4′‐Oxidation of Deoxyribose
  • Basic Protocol 2: Determination of 5′‐Oxidation of Deoxyribose
  • Basic Protocol 3: Determination of 3′‐Oxidation of Deoxyribose
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determination of 4′‐Oxidation of Deoxyribose

  Materials
  • 5′‐32P‐end‐labeled DNA or duplex oligonucleotide (e.g., see CPMB UNIT )
  • 1 mg/mL sonicated calf thymus DNA (unit 8.1)
  • 50 mM Tris⋅Cl ( appendix 2A), HEPES, or other buffer (not phosphate), containing 1 mM EDTA, pH 7
  • DNA‐damaging agent
  • 3 M sodium acetate, pH 7 ( appendix 2A)
  • 100% (v/v) ethanol, –20°C
  • 70% and 95% (v/v) ethanol
  • 1 M putrescine dihydrochloride (Prepare immediately before use)
  • 1 M aqueous hydrazine (Prepare immediately before use)
  • Formamide sequencing gel loading buffer without NaOH (e.g., see CPMB UNIT )
  • Maxam‐Gilbert sequencing markers prepared from the end‐labeled DNA (e.g., see CPMB UNIT )
  • 20% to 25% (w/v) polyacrylamide gel, 0.4 mm thick, containing urea and TBE electrophoresis buffer
  • Tris/borate/EDTA (TBE) electrophoresis buffer (see, e.g., CPMB UNIT )
  • Sequencing gel fixing solution (10% methanol, 10% acetic acid)
  • Sephadex G‐25 spin column (e.g., see CPMB UNIT )
  • Sequencing gel apparatus (30 × 40 cm) with 0.4 mm spacers and 2000 V power supply
  • Boiling water bath
  • Whatman 3MM paper, larger than the size of the gel
  • Sequencing gel drier, solvent trap, and vacuum pump (e.g., see CPMB UNIT )
  • X‐ray film or phosphor imager
CAUTION: Hydrazine is extremely toxic. It should be handled with gloves, in a fume hood, and disposed of properly. Methods of disposal may vary between different institutions. Consult with the institution's environmental safety office for the preferred means of storage and disposal of hydrazine.

Basic Protocol 2: Determination of 5′‐Oxidation of Deoxyribose

  Materials
  • 3′‐32P‐end‐labeled DNA or duplex oligonucleotide (e.g., see CPMB UNIT )
  • 1 M HEPES buffer, pH 7
  • 1 M sodium borohydride (prepare immediately before use)
  • 0.5 M piperidine (freshly prepared)
  • 8% (w/v) polyacrylamide gel, 0.4 mm thick, containing urea and TBE electrophoresis buffer
  • 90°C water bath
  • Additional reagents and equipment for determination of 4′‐oxidation of DNA (see protocol 1)

Basic Protocol 3: Determination of 3′‐Oxidation of Deoxyribose

  Materials
  • 0.1 M sodium chlorite in 20 mM potassium phosphate buffer, pH 4 (see appendix 2A for buffer)
  • 0.1 M sodium sulfite (Na 2SO 3)
  • Additional reagents and equipment as for 4′‐ and 5′‐oxidation of DNA (see Basic Protocols protocol 11 and protocol 22)
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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.
   Chin, D.‐H., Kappen, L.S., and Goldberg, I.H. 1987. 3′‐Formyl phosphate‐ended DNA: High energy intermediate in antibiotic‐induced DNA sugar damage. Proc. Natl. Acad.Sci. U.S.A. 84:7070‐7074.
   Christner, D.F., Frank, B.L., Kozarich, J.W., Stubbe, J., Golik, J., Doyle, T.W., Rosenberg, I.E., and Krishnan, B. 1992. Unmasking the chemistry of DNA cleavage by the esperamicins: Modulation of 4′‐hydrogen abstraction and bistranded damage by the fucose‐anthranilate moiety. J. Am. Chem. Soc. 114:8763‐8767.
   Dedon, P.C. and Goldberg, I.H. 1990. Sequence‐specific double‐strand breakage of DNA by neocarzinostatin within a staggered cleavagesite. J. Biol. Chem. 265:14713‐14716.
   Dedon, P.C. and Goldberg, I.H. 1992. Influence of thiol structure on neocarzinostatin activation and expression of DNA damage. Biochemistry 31:1909‐1917.
   Dedon, P.C., Jiang, Z.‐W., and Goldberg, I.H. 1992. Neocarzinostatin‐mediated DNA damage in a model AGT ACT site: Mechanistic studies of thiol‐sensitive partitioning of C4′ DNA damage products. Biochemistry 31:1917‐1727.
   Dedon, P.C., Salzberg, A.A., and Xu, J. 1993. Exclusive production of bistranded DNA damage by calicheamicin. Biochemistry 32:3617‐3622.
   Hangeland, J.J., De Voss, J.J., Heath, J.A., Townsend, C.A., Ding, W.‐D., Ashcroft, J.S., and Ellestad, G.A. 1992. Specific abstraction of the 5′(S)‐ and 4′‐deoxyribosyl hydrogen atoms from DNA by calicheamicin γ1I. J. Am. Chem. Soc. 114:9200‐9202.
   Henner, W.D., Rodriguez, L.O., Hecht, S.M., and Haseltine, W.A. 1983. Gamma ray induced deoxyribonucleic acid strand breaks: 3′ glycolate termini. J. Biol. Chem. 258:711‐713.
   Kappen, L.S., Goldberg, I.H., Frank, B.L., Worth, L.J., Christner, D.F., Kozarich, J.W., and Stubbe, J. 1991. Neocarzinostatin‐induced hydrogen atom abstraction from C‐4′ and C‐5′ of the T residue at a d(GT) step in oligonucleotides: Shuttling between deoxyribose attack sites based on isotope selection effects. Biochemistry 30:2034‐2042.
   Lindahl, T. and Andersson, A. 1972. Rate of chain breakage at apurinic sites in double‐stranded deoxyribonucleic acid. Biochemistry 11:3618‐3623.
   McGall, G., Rabow, L., Stubbe, J., and Kozarich, J.W. 1987. Incorporation of 18O into glycolic acid obtained from the bleomycin‐mediated degradation of DNA: Evidence for 4′‐radical trapping by 18O2. J. Am. Chem. Soc. 109:2836‐2837.
   Sitlani, A., Long, E.C., Pyle, A.M., and Barton, J.K. 1992. DNA photocleavage by phenanthrenequinone diimine complexes with rhodium (III): Shape‐selective recognition and reaction. J. Am. Chem.Soc. 114:2303‐2312.
   Steighner, R.J. and Povirk, L.F. 1990. Bleomycin‐induced DNA lesions at mutational hot spots: Implications for the mechanism of double‐strand cleavage. Proc. Natl. Acad. Sci. U.S.A. 87:8350‐8354.
   Stubbe, J. and Kozarich, J.W. 1987. Mechanisms of bleomycin‐induced DNA degradation. Chem. Rev. 87:1107‐1136.
   Xu, Y‐J., Zhen, Y.‐S., and Goldberg, I.H. 1994. C1027 chromophore, a potent new enediyne antitumor antibiotic, induces sequence‐specific double‐strand DNA cleavage. Biochemistry 33:5947‐5954.
   Yu, L., Golik, J., Harrison, R., and Dedon, P. 1994. The deoxyfucose‐anthranilate of esperamicin A1 confers intercalative DNA binding and causes a switch in the chemistry of bistranded DNA lesions. J. Am. Chem. Soc. 116:9733‐9738.
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