The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps.

Summary

The biological effects of energetic heavy ions are attracting increasing interest for their applications in cancer therapy and protection against space radiation. The cascade of events leading to cell death or late effects starts from stochastic energy deposition on the nanometer scale and the corresponding lesions in biological molecules, primarily DNA. We have developed experimental techniques to visualize DNA nanolesions induced by heavy ions. Nanolesions appear in cells as “streaks” which can be visualized by using different DNA repair markers. We have studied the kinetics of repair of these “streaks” also with respect to the chromatin conformation. Initial steps in the modeling of the energy deposition patterns at the micrometer and nanometer scale were made with MCHIT and TRAX models, respectively.

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.

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.

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.