Gisela Taucher-Scholz

Factors determining DNA double-strand break repair pathway choice in G2 phase.

DNA non‐homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double‐strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin‐associated DSBs (HC‐DSBs). Here, we examine the regulation of repair pathway usage at two‐ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation‐defective DNA‐PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP‐dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP‐1‐dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

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.

DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin

DNA double-strand breaks (DSBs) can induce chromosomal aberrations and carcinogenesis and their correct repair is crucial for genetic stability. The cellular response to DSBs depends on damage signaling including the phosphorylation of the histone H2AX (γH2AX). However, a lack of γH2AX formation in heterochromatin (HC) is generally observed after DNA damage induction. Here, we examine γH2AX and repair protein foci along linear ion tracks traversing heterochromatic regions in human or murine cells and find the DSBs and damage signal streaks bending around highly compacted DNA. Given the linear particle path, such bending indicates a relocation of damage from the initial induction site to the periphery of HC. Real-time imaging of the repair protein GFP-XRCC1 confirms fast recruitment to heterochromatic lesions inside murine chromocenters. Using single-ion microirradiation to induce localized DSBs directly within chromocenters, we demonstrate that H2AX is early phosphorylated within HC, but the damage site is subsequently expelled from the center to the periphery of chromocenters within ∼20 min. While this process can occur in the absence of ATM kinase, the repair of DSBs bordering HC requires the protein. Finally, we describe a local decondensation of HC at the sites of ion hits, potentially allowing for DSB movement via physical forces.

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.

DNA strand break induction and rejoining and cellular recovery in mammalian cells after heavy-ion irradiation

The induction of intracellular DNA strand breaks by X rays and various heavy charged particles was measured by the alkaline unwinding and alkaline and neutral filter elution techniques. No variations in strand break induction were found between the different cell lines under investigation. For a given particle, both the LET and the particle energy determined the efficiency to induce DNA lesions. RBE values for the total amount of induced strand breaks were always less than 1. For DNA double-strand breaks (DSBs), RBE values only slightly greater than 1 were determined for particle radiation with an LET around 300 keV/microns. Intracellular DSB/SSB ratios were found to be equivalent to data reported for in vitro systems using radioprotective conditions [Christensen et al., Int. J. Radiat. Biol. 22, 457-477, 1972; Taucher-Scholz et al., Adv. Space Res. 12(2-3), (2)73-(2)80, 1992]. Strand break rejoining as an indicator of cellular repair processes was detected even after high-LET irradiation (LET < or = 10,000 keV/microns). However, both the half-times of rejoining and the fraction of residual DNA breaks increased with the atomic number of the particle. After particle irradiation with LET values beyond 10,000 keV/microns, no rejoining of DNA strand breaks was found.

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.

Characterization of CDKN1A (p21) binding to sites of heavy-ion-induced damage: colocalization with proteins involved in DNA repair

Purpose : To determine an association of locally accumulated CDKN1A and DNA repair proteins at the sites of heavy-ion traversals. Materials and methods : CDKN1A, PCNA, DNA-PK, hMre11 and Rad50 were investigated for their subnuclear localization after irradiation with heavy-ions using immunocytochemical staining and confocal laser-scanning microscopy. Human fibroblasts (normal diploid or XPA, ATM- or NBS1-deficient lines and HPV16 E6-transfected cells) were used. Results : CDKN1A formed nuclear foci in G0/G1 normal human fibroblasts at the sites of particle traversal. Foci were persistent over hours and vanished after treatment with DNase-I. Formation of foci also occurred in NBS1- or ATM-deficient lines and in cells functionally abrogated for TP53. In normal fibroblasts, CDKN1A foci colocalized with particle-induced foci of the hMre11 and Rad50 proteins. However, only CDKN1A relocalization was observed in irradiated NBS1 cells. PCNA foci temporarily colocalizing with CDKN1A were also detected in normal fibroblasts after exposure to heavy-ions. In contrast, no radiation-induced subnuclear relocalization was found for DNA-PK. Conclusions : CDKN1A foci arise rapidly at sites of localized DNA damage induced by heavy-ions and are associated with the chromatin. Evidence is provided that localization of CDKN1A to foci is not dependent on functional TP53 and occurs independently of the formation of the hMre11/Rad50/NBS1 complex. The data support a yet unknown role of CDKN1A in sensing or early processing of radiation-induced DNA lesions.

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.