Abstract
Ionizing radiation induces DNA double strand breaks (DSBs) which can lead to the formation of chromosome rearrangements through error prone repair. In mammalian cells the positional stability of chromatin contributes to the maintenance of genome integrity. DSBs exhibit only a small, submicron scale diffusive mobility, but a slight increase in the mobility of chromatin domains by the induction of DSBs might influence repair fidelity and the formation of translocations. The radiation-induced local DNA decondensation in the vicinity of DSBs is one factor potentially enhancing the mobility of DSB-containing chromatin domains. Therefore in this study we focus on the influence of different chromatin modifying proteins, known to be activated by the DNA damage response, on the mobility of DSBs. IRIF (ionizing radiation induced foci) in U2OS cells stably expressing 53BP1-GFP were used as a surrogate marker of DSBs. Low angle charged particle irradiation, known to trigger a pronounced DNA decondensation, was used for the defined induction of linear tracks of IRIF. Our results show that movement of IRIF is independent of the investigated chromatin modifying proteins like ACF1 or PARP1 and PARG. Also depletion of proteins that tether DNA strands like MRE11 and cohesin did not alter IRIF dynamics significantly. Inhibition of ATM, a key component of DNA damage response signaling, resulted in a pronounced confinement of DSB mobility, which might be attributed to a diminished radiation induced decondensation. This confinement following ATM inhibition was confirmed using X-rays, proving that this effect is not restricted to densely ionizing radiation. In conclusion, repair sites of DSBs exhibit a limited mobility on a small spatial scale that is mainly unaffected by depletion of single remodeling or DNA tethering proteins. However, it relies on functional ATM kinase which is considered to influence the chromatin structure after irradiation.
Highlights
DNA double strand breaks (DSBs) arise from natural cellular processes as well as from external damaging agents like ionizing radiation and represent one of the most dangerous types of DNA lesions
The quantity of DSBs generated by an ion traversal scales with the linear energy transfer (LET) and is around 3–4 DSBs/mm for carbon ions and in the range of several hundred DSBs/mm for the heavier ions used in this study [21,28,29], whereby one mm roughly corresponds to the size of one 53BP1 focus. 53BP1 is a key regulator of DSB repair which accumulates at sites of DSBs in distinct foci colocalizing with cH2AX and persisting during ongoing repair [30]
The findings that neither MRE11 nor cohesin influences movement of DSBs suggests that not the connection of broken DNA strands but more likely changes in the surrounding chromatin structure largely define the dynamic behavior of DSBs on the time scale of minutes to hours. Mobility of both DSBs and undamaged chromatin has been described in yeast and in mammalian cells using a variety of approaches
Summary
DNA double strand breaks (DSBs) arise from natural cellular processes as well as from external damaging agents like ionizing radiation and represent one of the most dangerous types of DNA lesions. In HR DSBs are repaired correctly by the use of the undamaged homologous sequence as a template, whereas NHEJ fuses broken DNA ends together, a process which can lead to chromosome exchanges especially if multiple breaks are present. In this case it is yet unclear what promotes the joining of DSB ends, but proximity and movement of the ends seem to play an important role [5]. A higher mobility of damaged chromatin compared to non damaged chromatin was observed after induction of DSBs in yeast, most likely to facilitate homology search [9]
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