Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells |
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Authors: | LHF Mullenders AA Van Zeeland AT Natarajan |
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Institution: | 1. Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, Wassenaarseweg 72, 2333 AL Leiden The Netherlands;2. J.A. Cohen Institute, Interuniversity Institute for Radiopathology and Radiation Protection, Leiden The Netherlands |
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Abstract: | The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S1 of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop. |
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Keywords: | DNA repair Ultraviolet irradiation Nuclear matrix Repair patch distribution (Human fibroblast) |
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