Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in gamma-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay.
Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 microg/ml) and HeLa cells were irradiated by (60)Co gamma-rays, and abasic (AP) sites and endonuclease (Endo)III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10(6) base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10(6) bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10(6) bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation.
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan.
Mohammed
M
Mohsin Ali
Satofumi
S
Kurisu
Yoshihiro
Y
Yoshioka
Hiroaki
H
Terato
Yoshihiko
Y
Ohyama
Kihei
K
Kubo
Hiroshi
H
Ide
D020137:Base Pair Mismatch / Q000528:radiation effects; D001710:Biotin / Q000031:analogs & derivatives*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage; D004252:DNA Mutational Analysis / Q000379:methods*; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry* / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry* / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011829:Radiation Dosage
D029968:Escherichia coli Proteins; C074188:O-(biotinylcarbazoylmethyl)hydroxylamine; D001710:Biotin; D004247:DNA; C427203:calf thymus DNA; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli