Kvam E, Stokke T, Moan J, Steen H B
Department of Biophysics, Norwegian Radium Hospital, Montebello, Oslo.
Nucleic Acids Res. 1992 Dec 25;20(24):6687-93. doi: 10.1093/nar/20.24.6687.
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 degrees 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 microns 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)porphyrin, 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.
我们利用光敏剂的特性来研究细胞核中染色质包装的一个方面。荧光光敏剂中-四(3-羟基苯基)卟啉和光卟啉II均定位于核膜和其他膜结构中，但在细胞核内未发现。在1℃下，在存在敏化剂的情况下对细胞进行光照会诱导DNA双链断裂。DNA片段的长度分布通过脉冲场凝胶电泳确定。由于DNA损伤主要通过从敏化剂扩散小于0.1微米的单线态氧产生，因此推测DNA双链断裂是在核膜的这个距离内产生的。与此一致的是，随着光照时间的延长和敏化剂浓度的增加，DNA片段长度的分布达到一个平稳水平。相比之下，对于亲水性的核内敏化剂中-四(4-磺基苯基)卟啉，未发现这样的平稳水平。不同细胞类型的DNA片段长度的平稳分布具有相同的总体形状，平均片段长度范围为174至194千碱基对。在光裂解片段长度的广泛分布上发现了特定的基因，即c-myc、fos和p53。结果表明，在基因的每一侧，靠近核膜的染色质纤维的位置是随机变化的。
