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Nucleic Acids Research, 1992, Vol. 20, No. 24 6687-6693
© 1992

MOLECULAR BIOLOGY

Plateau distributions of DNA fragment lengths produced by extended light exposure of extranuclear photosensitizers in human cells

Egil Kvam, Trond Stokke, Johan Moan and Harald B. Steen

Department of Biophysics, Institute for Cancer Research, The Norwegian Radium Hospital Montebello, 0310 Oslo, Norway

Received August 14, 1992. Revised November 20, 1992. Accepted November 20, 1992.

We have exploited properties of photosensitizers to study an aspect of the packing of chromatin in the cell nucleus. The fluorescent photosensitizers mesotetra(3-hydroxyphenyl) porphyrin and Photofrin II were both localized in the nuclear membrane and other membrane structures, but could not be found inside the nuclei. Light exposure of cells at 1°C in the presence of the sensitizers induced DNA double-strand breaks. The length distributions of DNA fragments were determined by pulsed field gel electrophoresis. Because DNA damage is produced mainly via singlet oxygen diffusing less than 0.1 µm from the sensitizer, DNA double-strand breaks were supposedly produced within this distance of the nuclear membrane. Consistent with this, with prolonged illumination and with increasing concentrations of sensitizer the distribution of DNA fragment lengths reached a plateau level. In contrast, with the hydrophilic, intranuclear sensitizer meso-tetra(4-sulphonatophenyl)porphyrln, no such plateau level was found. The plateau distributions of DNA fragment lengths of different cell types had the same general shape with average fragment lengths ranging from 174 to 194 kilobasepairs. Particular genes, c-myc, fos and p53, were found on broad distributions of photocleaved fragment lengths. The results indicate that on each side of the genes the locus of the chromatin fibre situated close to the nuclear membrane, varied randomly.


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