Recruitment kinetics of DNA repair proteins Mdc1 and Rad52 but not 53BP1 depend on damage complexity.

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 T(0) after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ(1). Mdc1 accumulates faster (T(0) = 17 ± 2 s, τ(1) = 98 ± 11 s) than 53BP1 (T(0) = 77 ± 7 s, τ(1) = 310 ± 60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T(0) = 73 ± 16 s, τ(1) = 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 τ(1) 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

Parts of a micrograph time series of a U2OS cell nucleus showing GFP-tagged protein Mdc1.

<p>Irradiation took place on t  = 0. Foci formation can be observed already a few seconds after irradiation. Pairwise subtraction of the images reveals areas where foci are formed. The merge of these areas results in the region of interest (ROI) in which the foci brightness I_foci is evaluated for each image of the time series (cf. materials and methods). Scale bar in second image: 5 µm.</p

Foci intensity vs. time after irradiation.

<p>The relative foci intensity I_rel  =  I_foci/I_nucl of Mdc1 after irradiation with 5.2 Gy of 43 MeV carbon ions (A) and 4.8 Gy of 20 MeV protons (B) and 53BP1 after 7.6 Gy carbon (C) and 6.9 Gy proton irradiation (D) plotted for one cell and fitted with our model function (eq. 1). Irradiation took place on t  = 0. The insets show the protein accumulation after the irradiation with splayed time-axis.</p

53BP1 kinetics after ion irradiation compared to UV laser irradiation.

<p>53BP1 kinetics after ion irradiation compared to UV laser irradiation.</p

53BP1 kinetics.

<p>53BP1 kinetics (Hela pMC16-53BP1-GFP clone #2) after carbon and proton irradiation. Indicated are means and the standard errors of the means of 8–17 cells per data point. Because there was no significant difference between the various proton irradiations, pooled data are also shown in the last row.</p

Mdc1 kinetics.

<p>Mdc1 kinetics after carbon and proton irradiation for U2OS pEGFP-Mdc1 clone F1. Indicated are means and the standard errors of the means of 7–22 cells per sample.</p

LET and dose dependence of recruitment kinetics.

<p>Weighted mean values and standard errors of the means of the kinetics parameters T₀ and τ₁ of the proteins Mdc1 (A) and 53BP1 (B) after irradiation with 43 MeV carbon ions and two or three different doses of 20 MeV protons.</p

Dose and LET dependent kinetics of Rad52.

<p>10–20 min after irradiation with 43 MeV carbon ions (5.6 Gy, one ion per point) foci become visible. When applying the similar dose by 117 20 MeV protons per point first very weakly developed foci become visible not before three hours have elapsed (upper right nucleus). Increasing the number of protons per point accelerates the kinetics; with 512 protons per point (i.e. 24 Gy) already ten minutes after irradiation foci can be seen, so that kinetics is similar to that of 5.6 Gy carbon irradiation. Scale bars 20 µm.</p

Rad52 kinetics.

<p>Rad52 kinetics after carbon and proton irradiation of cell pool U2OS pEGFP-Rad52. Indicated are the times after irradiation until foci formation is visible by eye in microscopic images.</p