DNA supercoiling changes in nucleoids from irradiated L5178Y-S and -R cells.
DNA supercoiling ability was assayed following irradiation in two cell lines of differing radiosensitivity, L5178Y-S (LY-S) and L5178Y-R (LY-R). Cells treated with NaCl and Triton X-100 were exposed to increasing concentrations of the fluorescent, DNA-intercalating dye, propidium iodide (PI), and the diameter of the resulting fluorescent halo of DNA was measured. As the PI concentration was increased from 0.5 to 5 micrograms/ml, halo diameter increased from 20-25 to 45-55 microns due to the unwinding of the DNA supercoils. This process was similar for both cell lines under all conditions studied. As the PI concentration was increased to 50 micrograms/ml, the halo rewound to a diameter of 25-30 microns in unirradiated cells from both lines. However, following exposure to 3-12 Gy of 137Cs gamma rays, the ability of the DNA to be rewound was inhibited in a dose-dependent manner. Rewinding inhibition was greater in LY-S cells than in LY-R cells. Since the induction of DNA damage (e.g., single-strand DNA breaks) appears to be the same for both cell lines, this result implies that a similar extent of damage results in a greater loss of topological constraints on the DNA loops in LY-S. Such a change might be related to the protein composition of the nucleoid cores. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that nucleoids from LY-S cells were missing a 55-kDa protein present in LY-R.
Section of Cancer Biology, Mallinckrodt Institute of Radiology, Washington University, St. Louis, Missouri 63108.
M
M
Kapiszewska
W D
WD
Wright
C S
CS
Lange
J L
JL
Roti Roti
D000818:Animals; D002460:Cell Line; D004273:DNA, Neoplasm / Q000528:radiation effects*; D004278:DNA, Superhelical / Q000528:radiation effects*; D007940:Leukemia L5178 / Q000235:genetics* / Q000473:pathology; D007942:Leukemia, Experimental / Q000235:genetics*; D011419:Propidium; D011836:Radiation Tolerance
D004273:DNA, Neoplasm; D004278:DNA, Superhelical; D011419:Propidium