DNA is highly compacted by wrapping around histones and folding into higher order structures building chromatin. This chromatin can be subdivided in two classes: the highly transcriptional active, sparse euchromatin and the densely packed heterochromatin. The heterochromatin is transcriptionally inert but essential for the regulation of the gene expression and the entire architecture of the nucleus [1]. Here, we studied the DNA damage response within the heterochromatin. The distinct delimitation of euchromatin and heterochromatin in embryonic mouse fibroblasts, forming heterochromatic chromocenters, enabled us to clearly distinguish between these two chromatin fractions. For an aimed irradiation of the chromocenters with single ions we made use of the unique submicrometer resolution of the GSI microprobe.

The induction of localized DNA damage within a discrete nuclear volume is an important tool in DNA repair studies. Both charged particle irradiation and laser microirradiation (LMI) systems allow for such a localized damage induction, but the results obtained are difficult to compare, as the delivered laser dose cannot be measured directly. Therefore, we revisited the idea of a biological dosimetry based on the microscopic evaluation of irradiation-induced Replication Protein A (RPA) foci numbers. Considering that local dose deposition is characteristic for both LMI and charged particles, we took advantage of the defined dosimetry of particle irradiation to estimate the locally applied laser dose equivalent. Within the irradiated nuclear sub-volumes, the doses were in the range of several hundreds of Gray. However, local dose estimation is limited by the saturation of the RPA foci numbers with increasing particle doses. Even high-resolution 4Pi microscopy did not abrogate saturation as it was not able to resolve single lesions within individual RPA foci. Nevertheless, 4Pi microscopy revealed multiple and distinct 53BP1- and gamma H2AX-stained substructures within the lesion flanking chromatin domains. Monitoring the local recruitment of the telomere repeat-binding factors TRF1 and TRF2 showed that both proteins accumulated at damage sites after UVA-LMI but not after densely ionizing charged particle irradiation. Hence, our results indicate that the local dose delivered by UVA-LMI is extremely high and cannot be accurately translated into an equivalent ionizing radiation dose, despite the sophisticated techniques used in this study.

DNA is compacted into chromosomes in the nucleus of cells. The ends of these chromosomes (telomeres) are associated to proteins to form a dynamic cap that protects the DNA from being viewed as double-strand breaks (DSBs) and eliciting a DNA damage response. One of the proteins responsible for telomere capping is TRF2 (telomeric repeat binding factor 2). Recently this protein was also reported to function in homologous recombination repair of DSBs [1] and its recruitment to sites of laser microirradiation supporting an involvement in DNA repair was observed [2,3]. Here we demonstrate that 337nm-laser irradiation induces a fast recruitment of TRF2 and other telomere-associated proteins as TRF1[4] and TIN2 (TRF1-interacting nuclear protein 2), whereas densely ionizing charged particle irradiation give no indication of accumulation at sites of damaged DNA[4].

We studied the spatiotemporal organization of DNA damage processing by live cell microscopy analysis in human cells. In unirradiated U2OS osteosarcoma and HeLa cancer cells, a fast confined and Brownian-like motion of DNA repair protein foci was observed, which was not altered by radiation. By analyzing the motional activity of GFP-53BP1 foci in live cells up to 12-h after irradiation, we detected an additional slower mobility of damaged chromatin sites showing a mean square displacement of approximately 0.6 microm(2)/h after exposure to densely- or sparsely-ionizing radiation, most likely driven by normal diffusion of chromatin. Only occasionally, larger translational motion connected to morphological changes of the whole nucleus could be observed. In addition, there was no general tendency to form repair clusters in the irradiated cells. We conclude that long-range displacements of damaged chromatin domains do not generally occur during DNA double-strand break repair after introduction of multiple damaged sites by charged particles. The occasional and in part transient appearance of cluster formation of radiation-induced foci may represent a higher mobility of chromatin along the ion trajectory. These observations support the hypothesis that spatial proximity of DNA breaks is required for the formation of radiation-induced chromosomal exchanges.

Irradiation of cell nuclei with charged particles leads to the spatially defined production of DNA damage along the particle trajectories, thus facilitating studies on the dynamics of radiation-induced protein foci associated with lesion processing. Here we used visual inspection and computational analysis of the track morphology after immunodetection to describe the patterns of formation of gamma-H2AX foci and the repair-related proteins 53BP1 and RPA. We addressed the influence of lesion density on gamma-H2AX formation and the mobility of damaged chromatin sites by using low-angle irradiation of cell monolayers with low-energy carbon or uranium ions. We show the discrete formation of gamma-H2AX foci and the recruitment of repair-related proteins along ion trajectories over an LET range from 200 to 14300 keV/microm in human fibroblasts and in HeLa cells. The marked DSBs exhibited a limited mobility that was independent of the LET. The moderate extent of mobility in human fibroblasts pointed to a relatively stable positioning of the damaged chromatin domains during repair, in contrast to HeLa cells, which showed significant changes in the streak patterns in a fraction of cells, suggesting greater mobility in the local processing of DSBs. Our data indicate that the presence of single or multiple DSBs is not associated with an altered potential for movement of damaged chromatin. We infer that the repair of high-LET radiation-induced DSBs in mammalian cells is not coupled to an increased motional activity of lesions enhancing the probability of translocations.

Exposure of mammalian cell nuclei with charged particles leads to the spatially defined production of damaged chromatin domains along the particle trajectories. Analysis of track morphology facilitates studies on the dynamics of radiation-induced protein foci associated with lesion processing. Recently we described the discrete formation of γ-H2AX foci and the recruitment of repair related proteins along ion trajectories over a LET range from 200 to 14300 keV/μm in human fibroblasts and in HeLa cells (Fig. 1)[1].

In the last decade an increasing number of reports are published in which UV laser micro irradiation (UVLM) is used as a new tool to generate DNA double-strand breaks (DSBs) in localized regions within single nuclei [1]. Until then only heavy ions (HI) were known for their ability to induce DSBs in strictly localized areas of cell nuclei [2], but compared to UVLM systems the experimental handling is less comfortable. UVLM systems combine high magnification imaging with precise laser irradiation, thus, allowing the observation of early repair proteins in a sub-second timescale and a sub-micrometer resolution [3]. These advantages combined with the low costs compared to HI experiments make UVLM systems a tempting choice in the research field of DNA repair. Our aim is the characterisation of UVLM systems (λ = 337nm) by the comparison with HI irradiation. This work describes a comparative analysis of the induced DNA damage types after HI and UVLM, respectively.

The rise of laser micro irradiation (LMI) as a new tool to generate DNA double-strand breaks (DSBs) started in the late 90s, when this kind of systems showed the capability to irradiate specified sub-nuclear regions and to simultaneously observe fast and early damage responses. Although, a comparable experimental performance can also be realized with heavy ion (HI) irradiation, in comparison to LMI systems HI experiments have to face up their disadvantage of a much more complex setup. However - despite all advantages - one of the major drawbacks of LMI systems is the lack of any reliable measurement of the applied dose. The most promising approach to overcome this problem was described by Bekker-Jensen et al. (2006) who compared the amount of irradiation induced Replication Protein A (RPA) foci after LMI (λ = 337nm) and X-rays as a reference irradiation. The resulting dose effect curve for X-rays allows the allocation of the LMI data point at a nominal dose of 3Gy. Our results show that this holds true only if the foci are counted within the whole nucleus but varies strongly when the analyzed area (ROI) is reduced. While the homogenous dose distribution of X-rays results in a linear scaling of the foci number with respect to the area of the ROI, the number of RPA foci induced by LMI does not scale with the area of the ROI until the ROI is smaller than the irradiated region. The problem of a comparison of radiations with homogeneous and inhomogeneous dose distribution is also reflected when the method of Bekker-Jensen et al. (2006) is applied to HI irradiation. In this case even the dose of high LET Xenon ions (28Gy) is assigned to a nominal dose of about 2Gy. In conclusion, we were able to show that by shrinkage of the ROI the bias of inhomogeneities in the dose distribution of LMI and HI irradiation can be reduced and that the resulting dose effect curves of X-rays and HIs can be combined. Unfortunately, the laser data point is located in the saturation region of this dose effect curve (0.7 foci/µm²) making a unique allocation of the laser dose difficult. However, using this method, dose equivalents of locally applied laser irradiation is most probably in the order of a few hundred Gray within the irradiated volume.

Introduction DNA damage and in particular double-strand breaks (DSBs) pose a major threat to the preservation of genome integrity. Mammalian cells have developed efficient DNA repair mechanisms in response to DNA damage. In the non-homologous end-joining (NHEJ), one major pathway of DSB-repair, the DNA-dependent protein kinase (DNAPK) is supposed to participate in the early stages of DNA end detection and processing. The DNA-PK comprises the Ku subunits (Ku70 and Ku80) and the catalytic subunit DNAPKcs. The aim of this study was to visualize and analyse the recruitment of DNAPKcs to ionizing radiation induced DNA DSBs.