Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiationinduced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells. Ionizing radiation may produce cancer, death, and loss of neural function in humans and animals, and it may induce killing, mutation, and chromosomal aberrations in cells (1). Humans are exposed to low doses of radiation-during air travel, from radon in homes, during space travel, or in areas of low-level contamination, including former sites of nuclear weapon production-and can encounter much higher radiation doses in contaminated areas such as Chernobyl or during radiotherapy (1-5). However, ionizing radiation induces a plethora of types of DNA damages (6), and the identity of specific lesions responsible for the biological effects of radiation remains uncertain. Understanding the long-term effects of low and high doses of ionizing radiation on living organisms requires identification of critical radiation-induced DNA lesions, measurement of their repairability, and determination of the consequences of misrepaired or unrepaired persistent lesions.Lethal and mutagenic effects of ionizing radiation result principally from incompletely or incorrectly repaired DNA lesions (7,8). Ionizing radiation induces high levels of isolated DNA lesions, including SSBs, damaged bases, and abasic sites, located at a distance from other damages (6). Such isolated damages are generally repaired efficiently, and their repair may be increased by priming ionizing radiation doses (9).Ionizing radiation also induces closely spaced lesions, including double-strand breaks (DSBs)-two or more SSBs on opposing strands within about 10-20 bp (10, 11)-and has been postulated to produce other c...
Ionizing radiation induces both isolated DNA lesions and clustered damages-multiple closely spaced lesions (strand breaks, oxidized purines, oxidized pyrimidines, or abasic sites within a few helical turns). Such clusters are postulated to be difficult to repair and thus potentially lethal or mutagenic lesions. Using highly purified enzymes that cleave DNA at specific classes of damage and electrophoretic assays developed for quantifying isolated and clustered damages in high molecular length genomic DNAs, we determined the relative frequencies of total lesions and of clustered damages involving both strands, and the composition and origin of such clusters. The relative frequency of isolated vs clustered damages depends on the identity of the lesion, with approximately 15-18% of oxidized purines, pyrimidines, or abasic sites in clusters recognized by Fpg, Nth, or Nfo proteins, respectively, but only about half that level of frank single strand breaks in double strand breaks. Oxidized base clusters and abasic site clusters constitute about 80% of complex damages, while double strand breaks comprise only approximately 20% of the total. The data also show that each cluster results from a single radiation (track) event, and thus clusters will be formed at low as well as high radiation doses.
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