Dynamics of DNA-repair factors and chromosomes studied by laser-UVA-microirradiation and laser-photobleaching

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.