Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics

Abstract

The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp-5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high-LET alpha particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9+/-0.5 h and a slow component with a half-life of 16+/-9 h. For alpha particles, a fast component with a half-life of 0.7+/-0.4 h and a slow component with a half-life of 12+/-5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37+/-0.12) relative to low-LET radiation (0.22+/-0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites.