FRAP curves of MDC1 binding after charged particle irradiation.

<p>The mobility of MDC1 is drastically reduced at damaged DNA. MDC1 mobility is not only reduced at damaged sites but also in the whole nucleus when very high damaged densities are generated after heavy charged particle irradiation. Error bars are 95% confidence interval.</p

Schematic of interactions in our minimal model. MRN binds directly to the DSB strand ends.

<p>ATM is activated there and subsequently phosphorylates H2AX. MDC1 must be recruited to γH2AX before MRN can bind in the outer focus. In a final step, ATM also binds to recruited MDC1. For clarity, only the nucleosomes that contain H2AX are depicted.</p

Theoretical free diffusion constants D_calc for GFP-tagged NBS1 and MDC1 proteins estimated from the diffusion constant for GFP and the mass difference between pure GFP and the tagged proteins as well as experimental effective diffusion constants D_eff from experimental FRAP curves.* For free GFP D_calc = D_eff.

<p>Theoretical free diffusion constants D_calc for GFP-tagged NBS1 and MDC1 proteins estimated from the diffusion constant for GFP and the mass difference between pure GFP and the tagged proteins as well as experimental effective diffusion constants D_eff from experimental FRAP curves.* For free GFP D_calc  =  D_eff.</p

MDC1 protein accumulation at ion tracks.

<p>A: Normalized MDC1 protein recruitment to DNA damage sites after C- and Au-particle irradiation. Like NBS1, MDC1 accumulates faster at very high damage densities. Error bars are 95 % confidence interval. B: Monoexponential time constant, representing the time when 63% of the final foci intensity is reached (green lines in A), for MDC1 accumulation plotted as function of the LET. MDC1 protein accumulation accelerates with increasing LET, but saturates at higher LET values above 9000 keV/µm. Error bars are 95% confidence interval.</p

FRAP measurement of repair proteins bound at damaged DNA.

<p>U2OS cells expressing NBS1-GFP were irradiated with Ti ions (LET ∼270 keV/µm) under a low angle resulting in a streak-shaped foci pattern along the ion trajectory (red arrow). At time 0 s the fluorescence tag of the proteins in a small part of the streak are bleached with a short and intense laser pulse (cyan arrow). Fluorescence recovery in the bleached region represents the protein exchange at the DNA damage. Selected time frames are shown. Time labels correspond to the time after bleaching.</p

NBS1 binding at damaged DNA following CK2 inhibition.

<p>NBS1 binding at damaged DNA after CK2 inhibition preventing the interaction between NBS1 and MDC1. Cells were irradiated with Ar-ions. Error bars are standard deviation.</p

NBS1 protein accumulation at DSBs after ion irradiation.

<p>A: Normalized protein accumulation of NBS1 at DNA damage sites after C and V ion irradiation. When very high damage densities are created after exposure to a higher linear energy transfer (LET) radiation, NBS1 accumulates faster and saturates after shorter time. Error bars are 95% confidence interval. B: Monoexponential time constant, representing the time when 63% of the final foci intensity is reached (green lines in A), for NBS1 recruitment plotted as a function of the LET. Each LET value corresponds to one ion species. With increasing LET, the NBS1 accumulation accelerates up to about 3000 keV/µm and remains constant at further increasing ionization densities. Time constants of Rad50 and MRE11 accumulation are shown in red and blue respectively. Error bars are 95% confidence interval.</p

Influence of LET and CK2 inhibition on NBS1 binding to IRIF.

<p><b>A) NBS1 dissociation constant koff versus the LET.</b> Values were obtained by fitting the FRAP curves with the diffusion reaction model described by Sprague and coworkers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057953#pone.0057953-Sprague1" target="_blank">[40]</a>. As the LET increases, protein binding consta<i>n</i>ts approach the values of NBS1 binding obtained with CK2 inhibition. Error bars correspond to the asymptotic standard error. <b>B</b>) <b>Influence of CK2 inhibition on NBS1 and MDC1 foci size.</b> Immunofluorescence staining of NBS1 and MDC1 after Au ion irradiation with and without CK2-inhibition. U2OS cells were fixed 10 min after Au ion irradiation and immunocytochemically stained against NBS1 (green) and MDC1 (red). DNA was counterstained with DAPI (blue). Scalebar 10 µm.</p

FRAP measurements of NBS1 binding on damaged DNA after irradiation with ions of different LETs.

<p>NBS1 proteins bound to damaged DNA showed a reduced mobility compared to unbound proteins. For times beyond ∼10 s the FRAP curves showed a shallower increase with increasing LET. Error bars are not shown for the sake of clarity. Exemplary error bars are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057953#pone-0057953-g004" target="_blank">Figure 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057953#pone-0057953-g007" target="_blank">Figure 7</a>, the others are comparable.</p

Beamline microscopy of U2OS cells expressing NBS1-GFP.

<p>Cells were irradiated with Sm ions (LET 10290 keV/µm) at 0 s generating DNA damage along their trajectory. These damaged sites are detected by the repair protein NBS1 and the amount of accumulated protein increases with time. This causes the formation of clearly visible foci and a rise in the fluorescent signal over time. Only selected time-points are shown.</p