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.
Highlights
Delayed repair of clustered double-strand break (DSB) has been observed by immunostaining of markers of single strand breaks (SSBs), DSBs and base damage in mammalian cells[11]
To investigate the influence of simple and complex DNA lesions on the early DNA damage response (DDR) we made use of the different parts of the radiation tracks of HZE ions delivered by a particle accelerator in combination with real time live cell microscopy
Off-track DSBs produced by δ-electrons of these particles have been predicted using biophysical modelling[30], but hitherto have been studied only in fixed samples[31,32,33], unavoidably missing the early activation of repair factors and the dynamics of individual foci
Summary
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]. We measured the early protein recruitment at sites of simple and clustered, complex DSBs by live cell imaging. Individual foci in core and penumbra of the track were followed by live cell imaging from a few seconds up to 45 min post-irradiation. In this time frame we could visualise the early recruitment of repair factors and the release of the proteins after completion of repair in individual isolated and clustered DSBs. A similar setup was implemented for irradiation with X-rays using the same cells for comparison
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