Identification of the C4′-Oxidized Abasic Site as the Most Abundant 2-Deoxyribose Lesion in Radiation-Damaged DNA Using a Novel HPLC-Based Approach

A novel analytical high-performance liquid chromatography (HPLC)-based method of quantification of the yields of C4′-oxidized abasic sites, 1, in oxidatively damaged DNA has been elaborated. This new approach is based on efficient conversion of 1 into N-substituted 5-methylene-Δ3-pyrrolin-2-ones, 2, upon treatment of damaged DNA with primary amines in neutral or slightly acidic solutions with subsequent quantification of 2 by HPLC. The absolute and relative radiation-chemical yields of 1 in irradiated DNA solutions were re-evaluated using this method. The yields were compared with those of other 2-deoxyribose degradation products including 5-methylene-2(5H)-furanone, malondialdehyde, and furfural resulting from the C1′, C4′ and C5′-oxidations, respectively. The yield of free base release (FBR) determined in the same systems was employed as an internal measure of the total oxidative damage to the 2-deoxyribose moiety. Application of this technique identifies 1 as the most abundant sugar lesion in double-stranded (ds) DNA irradiated under air in solution (36% FBR). In single-stranded (ss) DNA this product is second by abundance (33% FBR) after 2-deoxyribonolactones (C1′-oxidation; 43% FBR). The production of nucleoside-5′-aldehydes (C5′-oxidation; 14% and 5% FBR in dsDNA and ssDNA, respectively) is in the third place. Taken together with the parallel reaction channel that converts C4′-radicals into malondialdehyde and 3′-phosphoglycolates, our results identify the C4′-oxidation as a prevalent pathway of oxidative damage to the sugar-phosphate backbone (50% or more of all 2-deoxyribose damages) in indirectly damaged DNA.

[1]  Marc M Greenberg,et al.  Probing DNA interstrand cross-link formation by an oxidized abasic site using nonnative nucleotides. , 2011, Bioorganic & medicinal chemistry.

[2]  M. Greenberg,et al.  Long patch base excision repair compensates for DNA polymerase β inactivation by the C4'-oxidized abasic site. , 2011, Biochemistry.

[3]  W. Bernhard,et al.  Factors Affecting the Yields of C1′ and C5′ Oxidation Products in Radiation-Damaged DNA: The Indirect Effect , 2010, Radiation research.

[4]  M. DeMott,et al.  Quantification of the 2-deoxyribonolactone and nucleoside 5'-aldehyde products of 2-deoxyribose oxidation in DNA and cells by isotope-dilution gas chromatography mass spectrometry: differential effects of gamma-radiation and Fe2+-EDTA. , 2010, Journal of the American Chemical Society.

[5]  Marc M Greenberg,et al.  Scope and mechanism of interstrand cross-link formation by the C4'-oxidized abasic site. , 2009, Journal of the American Chemical Society.

[6]  M. Fukuda,et al.  Photochemical generation of oligodeoxynucleotide containing a C4'-oxidized abasic site and its efficient amine modification: dependence on structure and microenvironment. , 2008, The Journal of organic chemistry.

[7]  J. Stubbe,et al.  GC/MS methods to quantify the 2-deoxypentos-4-ulose and 3'-phosphoglycolate pathways of 4' oxidation of 2-deoxyribose in DNA: application to DNA damage produced by gamma radiation and bleomycin. , 2007, Chemical research in toxicology.

[8]  Jean Cadet,et al.  Oxidation of the sugar moiety of DNA by ionizing radiation or bleomycin could induce the formation of a cluster DNA lesion , 2007, Proceedings of the National Academy of Sciences.

[9]  S. Dhar,et al.  Selective detection and quantification of oxidized abasic lesions in DNA. , 2007, Journal of the American Chemical Society.

[10]  M. Greenberg,et al.  Use of fluorescence sensors to determine that 2-deoxyribonolactone is the major alkali-labile deoxyribose lesion produced in oxidatively damaged DNA. , 2007, Angewandte Chemie.

[11]  Georges Lahoud,et al.  Aerobic fate of the C-3'-thymidinyl radical in single-stranded DNA. , 2006, Chemical research in toxicology.

[12]  M. Fukuda,et al.  Photochemical generation of C4'-oxidized abasic site containing oligodeoxynucleotide and its efficient amine modification. , 2006, Organic letters.

[13]  C. Sonntag.  Free-Radical-Induced DNA Damage and Its Repair: A Chemical Perspective , 2006 .

[14]  Clemens von Sonntag,et al.  Free-Radical-Induced DNA Damage and Its Repair , 2006 .

[15]  M. Roginskaya,et al.  2-Deoxyribonolactone lesions in X-ray-irradiated DNA: quantitative determination by catalytic 5-methylene-2-furanone release. , 2005, Angewandte Chemie.

[16]  P. Dedon,et al.  Chemical and Biological Evidence for Base Propenals as the Major Source of the Endogenous M1dG Adduct in Cellular DNA* , 2005, Journal of Biological Chemistry.

[17]  M. Roginskaya,et al.  The Release of 5-Methylene-2-Furanone from Irradiated DNA Catalyzed by Cationic Polyamines and Divalent Metal Cations , 2005, Radiation research.

[18]  P. Dedon,et al.  5'-(2-phosphoryl-1,4-dioxobutane) as a product of 5'-oxidation of deoxyribose in DNA: elimination as trans-1,4-dioxo-2-butene and approaches to analysis. , 2004, Chemical research in toxicology.

[19]  M. Greenberg,et al.  Repair of oxidized abasic sites by exonuclease III, endonuclease IV, and endonuclease III. , 2004, Biochemistry.

[20]  S. Hecht,et al.  Chemistry of the bleomycin-induced alkali-labile DNA lesion , 1999 .

[21]  H. Schuchmann,et al.  Bleomycin versus OH-radical-induced malonaldehydic-product formation in DNA. , 1999, International journal of radiation biology.

[22]  T. Tullius,et al.  Oxidative Strand Scission of Nucleic Acids: Routes Initiated by Hydrogen Abstraction from the Sugar Moiety. , 1998, Chemical reviews.

[23]  M. Kinter.  Analytical technologies for lipid oxidation products analysis. , 1995, Journal of chromatography. B, Biomedical applications.

[24]  John A. Murphy,et al.  Reactions of oxyl radicals with DNA. , 1995, Free radical biology & medicine.

[25]  M. Sevilla,et al.  Structure and Relative Stability of Deoxyribose Radicals in a Model DNA Backbone: Ab Initio Molecular Orbital Calculations , 1995 .

[26]  P. Dedon,et al.  Free-radical mechanisms involved in the formation of sequence-dependent bistranded DNA lesions by the antitumor antibiotics bleomycin, neocarzinostatin, and calicheamicin. , 1992, Chemical research in toxicology.

[27]  J. Stubbe,et al.  Identification and quantitation of the lesion accompanying base release in bleomycin-mediated DNA degradation , 1990 .

[28]  G. A. van der Marel,et al.  Chemistry of the alkali-labile lesion formed from iron(II) bleomycin and d(CGCTTTAAAGCG). , 1988, Biochemistry.

[29]  J. Gerlt,et al.  Identification of the alkaline-labile product accompanying cytosine release during bleomycin-mediated degradation of d(CGCGCG) , 1986 .

[30]  J. Stubbe,et al.  Mechanism of bleomycin: evidence for 4'-ketone formation in poly(dA-dU) associated exclusively with free base release. , 1985, Biochemistry.

[31]  J. Stubbe,et al.  Mechanism of bleomycin: evidence for a rate-determining 4'-hydrogen abstraction from poly(dA-dU) associated with the formation of both free base and base propenal. , 1985, Biochemistry.

[32]  N. Murugesan,et al.  STRUCTURE OF THE ALKALI‐LABILE PRODUCT FORMED DURING IRON(II)‐BLEOMYCIN‐MEDIATED DNA STRAND SCISSION , 1985 .

[33]  J. Stubbe,et al.  The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU). , 1983, The Journal of biological chemistry.

[34]  M. Dizdaroglu,et al.  Radiation Chemistry of DNA, II. Strand Breaks and Sugar Release by γ-Irradiation of DNA in Aqueous Solution. The Effect of Oxygen , 1975, Zeitschrift fur Naturforschung. Section C, Biosciences.

[35]  M. Dizdaroglu,et al.  STRAND BREAKS AND SUGAR RELEASE BY GAMMA-IRRADIATION OF DNA IN AQUEOUS SOLUTION , 1975 .

[36]  M. Dizdaroglu,et al.  Letter: Strand breaks and sugar release by gamma-irradiation of DNA in aqueous solution. , 1975, Journal of the American Chemical Society.

[37]  P. Dubs,et al.  Synthese von Azaprotoanemoninen , 1967 .