Clusters of DNA damage induced by ionizing radiation: formation of short DNA fragments. II. Experimental detection.
The basic 30-nm chromatin fiber in the mammalian cell consists of an unknown (possibly helical) arrangement of nucleosomes, with about 1.2 kb of DNA per 10-nm length of fiber. Track-structure considerations suggest that interactions of single delta rays or high-LET particles with the chromatin fiber might result in the formation of multiple lesions spread over a few kilobases of DNA (see the accompanying paper: W.R. Holley and A. Chatterjee, Radiat. Res. 145, 188-199, 1996). In particular, multiple DNA double-strand breaks and single-strand breaks may form. To test this experimentally, primary human fibroblasts were labeled with [3H]thymidine and exposed at 0 degrees C to X rays or accelerated nitrogen or iron ions in the LET range of 97-440 keV/microns. DNA was isolated inside agarose plugs and subjected to agarose gel electrophoresis under conditions that allowed good separation of 0.1-2 kb size DNA. The bulk of DNA remained in the well or migrated only a small distance into the gel. It was found that DNA fragments in the expected size range were formed linearly with dose with an efficiency that increased with LET. A comparison of the yield of such fragments with the yield of total DNA double-strand breaks suggests that for the high-LET ions a substantial proportion (20-90%) of DNA double-strand breaks are accompanied within 0.1-2 kb by at least one additional DNA double-strand break. It is shown that these results are in good agreement with theoretical calculations based on treating the 30-nm chromatin fiber as the target for ionizing particles. Theoretical considerations also predict that the clusters will contain numerous single-strand breaks and base damages. It is proposed that such clusters be designated "regionally multiply damaged sites." Postirradiation incubation at 37 degrees C resulted in a decline in the number of short DNA fragments, suggesting a repair activity. The biological significance of regionally multiply damaged sites is presently unknown.
Rydberg
B
B
Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley 94720, USA.
D002478:Cells, Cultured; D002843:Chromatin / Q000528:radiation effects* / Q000648:ultrastructure; D004247:DNA / Q000737:chemistry / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded; D004307:Dose-Response Relationship, Radiation; D006801:Humans; D007501:Iron; D018499:Linear Energy Transfer; D009584:Nitrogen; D011839:Radiation, Ionizing; D014965:X-Rays
D002843:Chromatin; D004277:DNA, Single-Stranded; D004247:DNA; D007501:Iron; D009584:Nitrogen