CLUSTERS OF DNA-DAMAGE INDUCED BY IONIZING-RADIATION - FORMATION OF SHORT DNA FRAGMENTS .2. EXPERIMENTAL DETECTION

The basic 30-nm chromatin fiber in the mammalian cell consists of an u nknown (possibly helical) arrangement of nucleosomes, with about 1.2 k b of DNA per 10-nm length of fiber. Track-structure considerations sug gest 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 partic ular, multiple DNA double-strand breaks and single-strand breaks may f orm. To test this experimentally, primary human fibroblasts were label ed with [H-3]thymidine and exposed at 0 degrees C to X rays or acceler ated nitrogen or iron ions in the LET range of 97-440 keV/mu m. DNA wa s isolated inside agarose plugs and subjected to agarose gel electroph oresis 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 dis tance into the gel. It was found that DNA fragments in the expected si ze range were formed linearly with dose with an efficiency that increa sed with LET. A comparison of the yield of such fragments with the yie ld of total DNA double-strand breaks suggests that for the high-LET io ns a substantial proportion (20-90%) of DNA double-strand breaks are a ccompanied within 0.1-2 kb by at least one additional DNA double-stran d break. It is shown that these results are in good agreement with the oretical calculations based on treating the 30-nm chromatin fiber as t he target for ionizing particles. Theoretical considerations also pred ict that the clusters will contain numerous single-strand breaks and b ase damages. It is proposed that such clusters be designated ''regiona lly multiply damaged sites.'' Postirradiation incubation at 37 degrees C resulted in a decline in the number of short DNA fragments, suggest ing a repair activity. The biological significance of regionally multi ply damaged sites is presently unknown. (C) 1996 by Radiation Research Society