Visualization of DNA loops in nucleoids from HeLa cells: assays for DNA damage and repair.
An assay for visualization of DNA loops undergoing supercoiling changes has been developed. The assay utilizes the fluorescent dye, propidium iodide (PI), which intercalates into the DNA and under the proper conditions causes the supercoiling status of the DNA to change. Thus, the DNA can be seen as a fluorescent halo that changes diameter with PI concentration. At low PI concentrations (0-7.5 micrograms/ml) the supercoils are relaxed with increasing PI, while at higher PI concentrations (7.50-50 micrograms/ml) supercoils in the opposite winding sense are rewound with increasing PI. When HeLa cells were irradiated with 1-20 Gy of 137Cs gamma-rays, the ability to rewind the DNA supercoils was inhibited in a dose-dependent manner, presumably because of the presence of radiation-induced DNA strand breakage, which removed the topological constraints on the DNA loops. These lesions were repaired rapidly during post-irradiation incubation. The ability of the DNA loops to be rewound was restored within 8 min after 10 Gy of gamma-irradiation, such that no difference from control cells could be detected. The half-time for repair of the radiation-induced lesions that inhibit DNA rewinding was similar to that for repair of DNA single strand breaks. The assay has certain advantages over current methods for assaying DNA damage in that it involves measurement of single cells and it does not require the DNA to be labeled with radioactive precursors.
Roti Roti
J L
JL
Section of Cancer Biology, School of Medicine, Washington University, St. Louis, Missouri 63108.
Wright
W D
WD
D004247:DNA / Q000528:radiation effects*; D004260:DNA Repair; D004278:DNA, Superhelical / Q000648:ultrastructure*; D005456:Fluorescent Dyes; D005720:Gamma Rays; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D011419:Propidium
D004278:DNA, Superhelical; D005456:Fluorescent Dyes; D011419:Propidium; D004247:DNA