Ionizing Radiation-induced DNA Strand Breakage and Rejoining in Specific Genomic Regions as Determined by an Alkaline Unwinding/Southern Blotting Method

Abstract

A recently developed, combined alkaline unwinding/Southern blotting assay was utilized to examine DNA damage and repair induced by ionizing radiation within specific large-scale genomic regions. Following treatment of MCF-7 breast tumour cells with 2-10-Gy γ-rays, strand breakage and rejoining were measured in bulk DNA, in the centromeric α-satellite region of chromosome 17, and in the chromatin regions containing the unexpressed β-globin gene and the expressed c-myc oncogene, which is known to be important for growth in the MCF-7 cell line. Damage in both the c-myc and β-globin regions was markedly greater than in either α-satellite or bulk DNA. However, the kinetics of strand break repair were approximately the same in c-myc as in α-satellite or bulk DNA. Surprisingly, the radiomimetic antibiotic bleomycin, which also induces free-radical-mediated strand breakage, showed considerably less heterogeneity of DNA damage among the genomic regions examined than did radiation. The results suggest that actively transcribed genes, as well as at least some inactive genes, are surrounded by large-scale domains of radiosensitive chromatin. With no apparent enhancement of rejoining, the increased incidence of strand breaks in these regions persists until rejoining is essentially complete. Changes in the integrity of specific chromatin regions may be an important aspect of DNA damage-induced cell death.