Open AccessVisualization of DNA loops in nucleoids from HeLa cells: Assays for DNA damage and repair
Author(s) -
Roti J. L. Roti,
Wright W. D.
Publication year - 1987
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990080505
Subject(s) - dna supercoil , nucleoid , dna , microbiology and biotechnology , dna damage , biophysics , dna repair , hela , biology , propidium iodide , chemistry , biochemistry , dna replication , in vitro , apoptosis , gene , escherichia coli , programmed cell death
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 μg/ml) the supercoils are relaxed with increasing PI, while at higher PI concentrations (7.50–50 μg/ml) supercoils in the opposite winding sense are rewound with increasing PI. When HeLa cells were irradiated with 1–20 Gy of 137 Cs γ‐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 γ‐irradiation, such that no difference from control cells could be detected. The halftime 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.