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Walter, Joachim (2003): Dynamics of DNA-repair factors and chromosomes studied by laser-UVA-microirradiation and laser-photobleaching. Dissertation, LMU München: Fakultät für Biologie

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Modern light microscopical techniques were employed to follow dynamical nuclear processes during the cell cycle and during DNA-repair. Laser-UVA-microirradiation The protein Rad51 is essential for the repair of double-strand breaks (DSBs) via the conservative homologous recombination repair pathway. To test the hypothesis that Rad51 localizes to damaged sites during DSB repair, a laser-UVA-microirradiation system was established. With this system spots with sizes around 1 µm in nuclei of living cells can be irradiated with UVA-light. After sensitization of cells by incorporation of BrdU into nuclear DNA and staining with the live cell dye Hoechst 33258, the system can be used to introduce double-strand breaks and single-strand breaks in the irradiated spots. The response of Rad51 to microirradiation By use of laser-UVA microirradiation the localization of Rad51 at damaged sites containing DNA double-strand breaks could be demonstrated. The accumulation of Rad51 at microirradiated sites was followed in cells fixed at increasing times after microirradiation. First Rad51 accumulations were visible 5 - 10 minutes after irradiation, and the number of cells with Rad51 accumulations increased until a plateau was reached 20 - 30 minutes after irradiation. In contrast, the majority of irradiated cells had accumulations of Mre11 protein already 5 - 10 minutes after irradiation. This is consistent with reports that nuclear Mre11 foci appeared early in the response to ionizing radiation, but absolute response times were faster after microirradiation than after ionizing radiation. Large-scale nuclear patterns were microirradiated, and Rad51 accumulations that reflected the shape of the irradiated patterns were found up to eight hours after irradiation. This conservation of the pattern of Rad51 accumulations, which reflect sites containing the damaged DNA, indicated that the chromatin in the irradiated cells performs no large scale reordering in response to DNA damage. The dynamics of chromosomes and chromosome territories In 1909 Theodor Boveri forwarded the hypothesis that arrangements of chromosome territories (CTs) are stably maintained during interphase, but subject to changes during mitosis. In the last decade several groups reported evidence for the stability of CT arrangements, but considerable movements of chromosomal subregions were also observed. The data concerning the maintenance or reordering of CTs during mitosis have been contradictory. Live cell imaging To follow the movements of chromosomes and CTs, a novel experimental approach was taken. Cells expressing a fusion protein of the core histone H2B with GFP (H2B-GFP) stably incorporate H2B-GFP into nucleosomes. In these cells chromatin regions were selectively marked by laser-photobleaching and followed by live cell microscopy. To this end, a live cell imaging system was established at a confocal laser-scanning microscope, which allows the observation of living cells for several days. Chromatin movements visualized by photobleached H2B-GFP To track possible movements in interphase cell nuclei, stripe patterns were bleached into nuclei at several stages of interphase. These patterns were retained for up to two hours, until they became invisible due to the replacement of bleached H2B-GFP by unbleached H2B-GFP, supporting the hypothesis that CT order is stably maintained during interphase. Nuclei, in which all chromatin except for a contiguous zone at one nuclear pole was bleached, were followed through mitosis. At prophase a number of unbleached chromosomal segments became visible. The segments showed a variable degree of clustering in metaphase. When daughter nuclei were formed, the segments locally decondensed into patches of unbleached chromatin. In all daughter cells the patches were separated by bleached chromatin, and clustered to a variable extent. These observations support the hypothesis that changes of chromosome neighborhoods occur during mitosis and that CT neighborhoods can profoundly vary from one cell cycle to the next.