Burkhard Jakob

Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

Biological Imaging of Heavy Charged-Particle Tracks

Abstract Jakob, B., Scholz, M. and Taucher-Scholz, G. Biological Imaging of Heavy Charged-Particle Tracks. Radiat. Res. 159, 676–684 (2003). The immunocytochemical response to DNA damage induced by low-energy bismuth and carbon ions was investigated in normal human fibroblasts. Inside the nuclei, the traversing charged particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation under a standard geometric setup with the beam direction perpendicular to the cell monolayer generates spots of these proteins as described previously for MRE11B (hMre11), CDKN1A (p21) and PCNA (Jakob et al., Int. J. Radiat. Biol. 78, 75–88, 2002). Here we present data obtained with a new irradiation geometry characterized by a small angle between the beam direction and the monolayer of cells. This new irradiation geometry leads to the formation of protein aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus facilitating the analysis of the fluorescence distributions along the particle trajectories. Measurements of fluorescence intensity along the ion tracks in double- and triple-stained samples revealed a strict spatial correlation for the occurrence of CDKN1A and MRE11B clusters. In addition, immunostained γ-H2AX is used as a marker of double-strand breaks (DSBs) to visualize the localized induction of these lesions along the particle paths. A clear coincidence of CDKN1A and γ-H2AX signals within the ion-induced streaks is observed. Also for PCNA, which mainly associates with lesions processed by excision repair, a strict colocalization with the MRE11B aggregations was found along the ion trajectories, despite the higher estimated yield of this type of lesions compared to DSBs. Strikingly similar patterns of protein clusters are generated not only for the various proteins studied but also using different ion species from carbon to bismuth, covering LET values ranging from about 300 to 13600 keV/μm and producing estimated DSB densities differing by a factor around 45. The patterns of protein clustering along the very heavy-ion trajectories appear far more heterogeneous than expected based on idealized DSB distributions arising from model calculations. The results suggest that additional factors like compaction or confined movement of chromatin are responsible for the observed clustering of proteins.

Autophosphorylation and ATM activation: Additional sites add to the complexity

The recognition and signaling of DNA double strand breaks involves the participation of multiple proteins, including the protein kinase ATM (mutated in ataxia-telangiectasia). ATM kinase is activated in the vicinity of the break and is recruited to the break site by the Mre11-Rad50-Nbs1 complex, where it is fully activated. In human cells, the activation process involves autophosphorylation on three sites (Ser367, Ser1893, and Ser1981) and acetylation on Lys3016. We now describe the identification of a new ATM phosphorylation site, Thr(P)1885 and an additional autophosphorylation site, Ser(P)2996, that is highly DNA damage-inducible. We also confirm that human and murine ATM share five identical phosphorylation sites. We targeted the ATM phosphorylation sites, Ser367 and Ser2996, for further study by generating phosphospecific antibodies against these sites and demonstrated that phosphorylation of both was rapidly induced by radiation. These phosphorylations were abolished by a specific inhibitor of ATM and were dependent on ATM and the Mre11-Rad50-Nbs1 complex. As found for Ser(P)1981, ATM phosphorylated at Ser367 and Ser2996 localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser367 and Ser2996 was functionally important because mutant forms of ATM were defective in correcting the S phase checkpoint defect and restoring radioresistance in ataxia-telangiectasia cells. These data provide further support for the importance of autophosphorylation in the activation and function of ATM in vivo.

Targeted Irradiation of Mammalian Cells Using a Heavy-Ion Microprobe

Abstract Heiß, M., Fischer, B. E., Jakob, B., Fournier, C., Becker, G. and Taucher-Scholz, G. Targeted Irradiation of Mammalian Cells Using a Heavy-Ion Microprobe. Radiat. Res. 165, 231–239 (2006). The existing focusing heavy-ion microprobe at the Gesellschaft für Schwerionenforschung in Darmstadt (Germany) has been modified to enable the targeted irradiation of single, selected cells with a defined number of ions. With this setup, ions in the range from helium to uranium with linear energy transfers (LETs) up to ∼15,000 keV/μm can be positioned with a precision of a few micrometers in the nuclei of single cells that are growing in culture on a thin polypropylene film. To achieve this accuracy, the microbeam traverses a thin vacuum window with minimal scattering. Electron emission from that window is used for particle detection. The cells are kept in a specially designed dish that is mounted directly behind the vacuum window in a setup allowing the precise movement and the imaging of the sample with microscopic methods. The cells are located by an integrated software program that also controls the rapid deflection and switching of the beam. In this paper, the setup is described in detail together with the first experiments showing its performance. We describe the ability of the microprobe to reliably hit randomly positioned etched nuclear tracks in CR-39 with single ions as well as the ability to visualize the ion hits using immunofluorescence staining for 53BP1 as a marker of DNA damage in the targeted cell nuclei.

Positional Stability of Damaged Chromatin Domains along Radiation Tracks in Mammalian Cells

Abstract Jakob, B., Splinter, J. and Taucher-Scholz, G. Positional Stability of Damaged Chromatin Domains along Radiation Tracks in Mammalian Cells. Radiat. Res. 171, 405–418 (2009). 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 γ-H2AX foci and the repair-related proteins 53BP1 and RPA. We addressed the influence of lesion density on γ-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 γ-H2AX foci and the recruitment of repair-related proteins along ion trajectories over an LET range from 200 to 14300 keV/μm 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.

Live Cell Imaging of Heavy-Ion-Induced Radiation Responses by Beamline Microscopy

Abstract Jakob, B., Rudolph, J. H., Gueven, N., Lavin, M. F. and Taucher-Scholz, G. Live Cell Imaging of Heavy-Ion-Induced Radiation Responses by Beamline Microscopy. Radiat. Res. 163, 681–690 (2005). To study the dynamics of protein recruitment to DNA lesions, ion beams can be used to generate extremely localized DNA damage within restricted regions of the nuclei. This inhomogeneous spatial distribution of lesions can be visualized indirectly and rapidly in the form of radiation-induced foci using immunocytochemical detection or GFP-tagged DNA repair proteins. To analyze faster protein translocations and a possible contribution of radiation-induced chromatin movement in DNA damage recognition in live cells, we developed a remote-controlled system to obtain high-resolution fluorescence images of living cells during ion irradiation with a frame rate of the order of seconds. Using scratch replication labeling, only minor chromatin movement at sites of ion traversal was observed within the first few minutes of impact. Furthermore, time-lapse images of the GFP-coupled DNA repair protein aprataxin revealed accumulations within seconds at sites of ion hits, indicating a very fast recruitment to damaged sites. Repositioning of the irradiated cells after fixation allowed the comparison of live cell observation with immunocytochemical staining and retrospective etching of ion tracks. These results demonstrate that heavy-ion radiation-induced changes in subnuclear structures can be used to determine the kinetics of early protein recruitment in living cells and that the changes are not dependent on large-scale chromatin movement at short times postirradiation.

Immediate Localized CDKN1A (p21) Radiation Response after Damage Produced by Heavy-Ion Tracks

Abstract Jakob, B., Scholz, M. and Taucher-Scholz, G. Immediate Localized CDKN1A (p21) Radiation Response after Damage Produced by Heavy-Ion Tracks. Using confocal microscopy on immunofluorescence-stained cells, we have investigated the response of CDKN1A (p21), one of the key proteins involved in the DNA damage response pathway, after irradiation with accelerated lead or chromium ions. Each traversal of an accelerated ion leads to the formation of a single, bright focus of the CDKN1A protein in the nuclei of human fibroblasts within 2 min after irradiation at 4°C. This immediate, localized CDKN1A response is specific for particle irradiation with a high linear energy transfer (LET), whereas X irradiation, after a period of induction, yields a diffusely spread pattern, in line with the differences in the microscopic dose deposition pattern of both radiation types. The particle-induced CDKN1A foci persist for several hours until they become diffuse and vanish. These findings suggest that CDKN1A accumulates at the sites of primary DNA damage, possibly mediated by the interaction with proteins involved in DNA repair. Here, for the first time, an immediate biological response confined to the radial extension of low-energy particle tracks (∼1 μm) is directly visualized and correlated to ion traversals. This indicates that particle irradiation represents an ideal tool to study the processing of biological damage induced in defined subnuclear regions.

ATM protein-dependent phosphorylation of Rad50 protein regulates DNA repair and cell cycle control

The Mre11/Rad50/NBN complex plays a central role in coordinating the cellular response to DNA double-strand breaks. The importance of Rad50 in that response is evident from the recent description of a patient with Rad50 deficiency characterized by chromosomal instability and defective ATM-dependent signaling. We report here that ATM (defective in ataxia-telangiectasia) phosphorylates Rad50 at a single site (Ser-635) that plays an important adaptor role in signaling for cell cycle control and DNA repair. Although a Rad50 phosphosite-specific mutant (S635G) supported normal activation of ATM in Rad50-deficient cells, it was defective in correcting DNA damage-induced signaling through the ATM-dependent substrate SMC1. This mutant also failed to correct radiosensitivity, DNA double-strand break repair, and an S-phase checkpoint defect in Rad50-deficient cells. This was not due to disruption of the Mre11/Rad50/NBN complex revealing for the first time that phosphorylation of Rad50 plays a key regulatory role as an adaptor for specific ATM-dependent downstream signaling through SMC1 for DNA repair and cell cycle checkpoint control in the maintenance of genome integrity.

Biological dose estimation of UVA laser microirradiation utilizing charged particle-induced protein foci

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.

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1.