Differential Repair Protein Recruitment at Sites of Clustered and Isolated DNA Double-Strand Breaks Produced by High-Energy Heavy Ions

Abstract

DNA double-strand break (DSB) repair is crucial to maintain genomic stability. The fidelity of the repair depends on the complexity of the lesion, with clustered DSBs being more difficult to repair than isolated breaks. Using live cell imaging of heavy ion tracks produced at a high-energy particle accelerator we visualised simultaneously the recruitment of different proteins at individual sites of complex and simple DSBs in human cells. NBS1 and 53BP1 were recruited in a few seconds to complex DSBs, but in 40% of the isolated DSBs the recruitment was delayed approximately 5 min. Using base excision repair (BER) inhibitors we demonstrate that some simple DSBs are generated by enzymatic processing of base damage, while BER did not affect the complex DSBs. The results show that DSB processing and repair kinetics are dependent on the complexity of the breaks and can be different even for the same clastogenic agent. Repair of DNA double-strand breaks is a necessary pathway to maintain genomic stability in mammalian cells 1. The DNA damage response (DDR) signalling cascade is rapid and hierarchically coordinated, with many proteins being recruited to the damage sites at different times and depending on the repair pathway. Complex DSBs, i.e. complex lesions involving multiple strand breaks and/or oxidative damages within two helical turns 2,3 , are more difficult to repair than isolated, frank DSBs 4. Complex DSBs can be produced by ionising radiation (IR) 3,5 , and their fraction and complexity and clustering increases for densely ionising, high-linear energy transfer (LET) radiation such as α-particles and heavy ions 6. Endogenous DSBs (simple DSBs produced during replication) have much higher frequency than IR-induced DSBs at low doses, but the latter include complex DSBs 7. These complex lesions are considered ultimately responsible for the late effects of low doses of IR 8 , including environmental exposures and cosmic radiation risk in space travel. The repair of simple and complex DSBs is therefore crucial to understand the difference between endogenous and exogenous genotoxicity and for modelling the risk related to exposure to low doses IR on Earth 9 and in space 10. Delayed repair of clustered DSBs has been observed by immunostaining of markers of single strand breaks (SSBs), DSBs and base damage in mammalian cells 11. Here we measured the early protein recruitment at sites of simple and clustered, complex DSBs by live cell imaging. Immunostaining on fixed samples is unable to identify the early kinetics and cannot follow the evolution of individual foci, therefore providing only average values 12. High-charge and-energy (HZE) ions offer a unique opportunity for these studies as they simultaneously produce both clustered (along the track) and isolated (off-track) DSBs. In fact, the inner part of the ion track (core) includes primary particle ionizations and low-energy electrons, and results mostly in complex, clustered DSBs for HZE ions at high linear energy transfer (LET), whereas low-LET high-energy ionised ...

Correlative Light and Electron Microscopy (CLEM) Analysis of Nuclear Reorganization Induced by Clustered DNA Damage Upon Charged Particle Irradiation

“…Fluorescence imaging of samples performed directly after the different types of fixation revealed the characteristic formation of radiation-induced foci (IRIF) indicated by the accumulation of 53BP1-GFP fluorescence at the DNA damaged domains after irradiation with C or Fe ions ( Figure 1B,D, Figure 2B,D,F,H). The characteristics of the 53BP1 IRIF were in full accordance with live cell observations before fixation and with the ones obtained previously after particle irradiation [26,27]. The finding that this distinct fluorescence pattern, as well as sufficient signal intensity, was preserved after fixation indicated that both fixation protocols did not affect the 53BP1 distribution and the nuclear architecture at the detectable level.…”

Ionizing Radiation and Complex DNA Damage: Quantifying the Radiobiological Damage Using Monte Carlo Simulations

“…Accumulating evidence, as presented in a recent review article by Mavragani et al [127], shows that high-LET radiation usually triggers a very different biological response compared to low-LET IR like X-rays or γ-rays. Recent data by Jacob et al suggests that even within the ion track core and the off-track δ-rays (high-energy electrons), there is a substantial difference in the complexity of damage, for example DSBs [128]. The core DNA lesions are difficult to repair and show a delay compared to the off-track δ-ray induced DSBs which rather resemble in complexity the ones induced by X-rays.…”

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

“…Using automatic extraction of RIF imaging features and linear programming techniques, this technique enabled to characterize detailed RIF kinetics for 24 h prior to and after exposure to low and high doses of ionizing radiation, showing that DNA repair occurs in a limited number of large repair domains. In an impressive recent work [80], Jacob et al performed live cell imaging on heavy ion irradiated cells expressing NBS1-GFP or 53BP1-GFP or EGFP-XRCC1. To achieve live cell imaging at early time points post-irradiation (a few seconds), the microscope was placed in the beam line and controlled remotely.…”