Irradiation damage in chromatin isolated from V-79 Chinese hamster lung fibroblasts.
The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl2. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure.
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000032:analysis / Q000528:radiation effects* / Q000648:ultrastructure; D006224:Cricetinae; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D004996:Ethidium; D006657:Histones / Q000032:analysis / Q000502:physiology; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009707:Nucleosomes / Q000032:analysis; D014965:X-Rays
D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D015636:Magnesium Chloride; D004996:Ethidium; D008274:Magnesium