Recruitment Kinetics of DNA Repair Proteins Mdc1 and Rad52 but Not 53BP1 Depend on Damage Complexity

Abstract

The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET = 2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T0 after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ1. Mdc1 accumulates faster (T0 = 17±2 s, τ1 = 98±11 s) than 53BP1 (T0 = 77±7 s, τ1 = 310±60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T0 = 73±16 s, τ1 = 1050±270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ1 of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.

Nuclear dynamics of radiation-induced foci in euchromatin and heterochromatin

“…Typically, the presence of multiple RIFs results in a brighter signal, which is detected faster and persists longer. For example, γH2AX RIF are brighter after exposure to high-LET than they are after exposure to X-rays [11]; similar results have also been observed for Mdc1, Rad52 and 53BP1 [40, 86, 90]. We initially thought this was because a stronger response is necessary to repair more complex breaks.…”

“…We initially thought this was because a stronger response is necessary to repair more complex breaks. However, 53BP1 protein diffusion measurements in time-lapse studies revealed that 53BP1 proteins are not recruited to RIF faster after high-LET [40, 90]. This suggests that RIF are formed at the same rate independent of dose or break complexity, and that clustering is instead responsible for brighter, larger and earlier appearance of RIF at higher doses of IR.…”

Chromatin Compaction Protects Genomic DNA from Radiation Damage

“…Although these in vivo results reflect many indirect effects, making it difficult to draw simple conclusions, they appear to be in good agreement with our in vitro findings. For further in vivo study, the use of an ion microbeam [57] or UV laser microirradiation [58] would be useful, since both allow the targeted irradiation of heterochromatic and euchromatic nuclear areas and may enable a direct comparison of the generation of DSBs in these compartments either by a TUNEL assay or the identification of γH2AX.…”

SIRT 7 promotes genome integrity and modulates non‐homologous end joining DNA repair

“…3D reconstruction and image analysis were performed using Imaris software (Bitplane, A.G.). Microirradiation experiments and quantification of recruitment kinetics were performed following methods previously described (Hable et al , 2012) with some modifications. FRAP experiments were carried out as previously described (Bosch‐Presegue et al , 2011).…”