Characterization and mechanism of formation of tandem lesions in DNA by a nucleobase peroxyl radical.
5,6-Dihydro-2'-deoxyuridin-6-yl (1) was independently generated via photolysis of 3. The radical is an analogue of the major reactive species produced from thymidine upon reaction with hydroxyl radical, which is the dominant DNA-damaging agent produced by the indirect effect of gamma-radiolysis. Under aerobic conditions, the peroxyl radical (2) derived from 1 reacts approximately 82% of the time with either the 5'- or 3'-adjacent nucleotide to produce two contiguously damaged nucleotides, known as tandem lesions. The structures and distribution of tandem lesions were investigated using probes that selectively detect abasic sites, ESI-MS/MS, and competition kinetics. In addition to 2-deoxyribonolactone, nonoxidized abasic sites were detected. 18O-Labeling verified that H2O was the source of oxygen in the abasic sites, but that O2 was the source of the oxygen in the 5,6-dihydro-6-hydroxy-2'-deoxyuridine derived from 2. ESI-MS/MS experiments, in conjunction with isotopic labeling, identified several products and provided direct evidence for peroxyl radical addition to the adjacent thymine bases. Kinetic studies revealed that peroxyl radical addition to the 5'-thymine was favored by approximately 4-5-fold over C1'-hydrogen atom abstraction from the respective deoxyribose ring, and that 2-deoxyribonolactone formation accounts for approximately 11% of the total amount of tandem lesions produced. These results suggest that tandem lesions, whose biochemical effects are largely unknown, constitute a major family of DNA damage products produced by the indirect effect of gamma-radiolysis.
Hong
In Seok
IS
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Carter
K Nolan
KN
Sato
Kousuke
K
Greenberg
Marc M
MM
D000468:Alkalies; D004247:DNA / Q000737:chemistry*; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D010545:Peroxides / Q000737:chemistry*; D010782:Photolysis; D021241:Spectrometry, Mass, Electrospray Ionization; D013400:Sugar Acids / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D053719:Tandem Mass Spectrometry
C479982:5,6-dihydro-2'-deoxyuridin-6-yl; D000468:Alkalies; D010545:Peroxides; D013400:Sugar Acids; D013438:Sulfhydryl Compounds; C049375:perhydroxyl radical; C056405:2,4,5-trihydroxypentanoic acid gamma-lactone; D004247:DNA; D003857:Deoxyuridine