Spin-trapping detection of precursors of hydroxyl-radical-induced DNA damage: identification of precursor radicals of DNA strand breaks in oligo(dC)10 and oligo(dT)10.
A spin-trapping method combined with enzymatic digestion and high-performance liquid chromatography was employed to detect hydroxyl-radical-induced precursors of strand breaks in oligonucleotides ((dC)10 and (dT)10) as DNA models. Radicals produced as precursors of both strand breaks and base alterations were first trapped by the spin trap 2-methyl-2-nitrosopropane. The oligonucleotides containing spin adducts were subsequently digested by snake venom phosphodiesterase to release low-molecular-weight nitroxide fragments. In this way, several spin adducts were separated by high-performance liquid chromatography. In both oligonucleotides, ESR spectra attributable to the spin adducts derived from trapping of a precursor radical of strand breaks (the C4'-sugar radical) were observed. To further confirm this assignment, the induction of strand breaks was examined by polyacrylamide gel electrophoresis of 5'-32P-end-labeled oligonucleotides. Autoradiograms of the gels showed that the fragments corresponding to monomers to 9mers were formed in both oligonucleotides. When experiments were carried out under conditions in which hydroxyl radicals reacted with oligomers in the presence of the spin trap, the spin trap was found to suppress the fragmentation more than it did by scavenging hydroxyl radicals, indicating that the precursor radical of strand breaks (the C4' radical) was trapped. The present experiments showed that the spin-trapping method combined with gel electrophoresis was a good approach to identify sites of radical damage which cause strand breaks in oligonucleotides (probably in DNA).
Department of Radiation Biology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
M
M
Kuwabara
H
H
Ohshima
F
F
Sato
A
A
Ono
A
A
Matsuda
D004249:DNA Damage; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D017665:Hydroxyl Radical; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000302:isolation & purification; D013113:Spin Labels
D009838:Oligodeoxyribonucleotides; D013113:Spin Labels; C027903:oligo (dT); C033539:oligodeoxycytidylic acid; D017665:Hydroxyl Radical