Knockdown of SMC1 or MRE11 does not influence mobility of IRIF.

<p>A) Mean square displacement (msd) of 53BP1 foci after irradiation with Pb (LET: 13500 keV/μm) is plotted against time for wt (blue line), SMC1 knockdown (red line) and MRE11 knockdown cells (green line). B) Western blots of U2OS cells 48 h after knockdown of SMC1 and MRE11 with tubulin as loading control.</p

Influence of repair-related chromatin modifying proteins on mobility of 53BP1-GFP foci in irradiated U2OS cells.

<p>A) Plot of the mean square displacement (msd) of IRIF in control cells (wt) and cells which were depleted for ATP 30 min prior to carbon ion (LET: 170 keV/μm) irradiation (n = 7). Errors represent SEM in all plots. B) Msd of IRIF in cells after knockdown of ACF1 (n = 23) and non treated controls (wt) (n = 11) after irradiation with Cr (LET: 2360 keV/μm). C) Comparison of the msd of IRIF in cells after inhibition of PARP (10 μM PJ34) and controls (wt) (n = 15). Cells were irradiated with C (LET: 170 keV/μm). D) Msd of IRIF in cells after knockdown of PARG (n = 15) and in non treated controls (wt) (n = 11). Cells were irradiated with Cr (LET: 2360 keV/μm). E) Western Blot showing the ACF1 and PARG knockdown efficiency with actin as loading control. F) U2OS cells treated with 20 mM H₂O₂ for 10 min, fixed and stained for PAR (green) and DNA (blue) show efficiency of PARP1 inhibition with 10 μM PJ34.</p

Inhibition of ATM constricts mobility of 53BP1 foci induced by heavy ion or photon irradiation.

<p>Irradiation of U2OS cells was performed by Cr (LET: 2630 keV/μm) for plots A and C and by 1 Gy X-rays for plots B and D. The mean square displacement (msd) of IRIF is plotted over time. Errors represent SEM. <b>A, B</b>) Msd plots of control (solid squares) (Cr n = 11, X-ray n = 21) and ATM inhibited cells (KU55933 open squares) (Cr n = 31, X-ray n = 11) fitted for subdiffusion (red line) and confined diffusion (blue line). <b>C,D</b>) Bar graphs of the average msd after 100 min observation time by live cell microscopy for control and ATM inhibited cells (KU55933) after irradiation with Cr <b>C</b>) and after irradiation with 1 Gy X-rays <b>D</b>). <b>E</b>) U2OS-53BP1-GFP cells irradiated with 1 Gy X-rays, fixed after 30 min and stained for pATM (red) and DNA (blue). Wt compared to cells treated with 15 μM KU55933 for 2 hours (ATMi) show efficiency of ATM kinase inhibition.</p

DNA end resection is needed for the repair of complex lesions in G1-phase human cells

<div><p>ABSTRACT</p><p>Repair of DNA double strand breaks (DSBs) is influenced by the chemical complexity of the lesion. Clustered lesions (complex DSBs) are generally considered more difficult to repair and responsible for early and late cellular effects after exposure to genotoxic agents. Resection is commonly used by the cells as part of the homologous recombination (HR) pathway in S- and G2-phase. In contrast, DNA resection in G1-phase may lead to an error-prone microhomology-mediated end joining. We induced DNA lesions with a wide range of complexity by irradiation of mammalian cells with X-rays or accelerated ions of different velocity and mass. We found replication protein A (RPA) foci indicating DSB resection both in S/G2- and G1-cells, and the fraction of resection-positive cells correlates with the severity of lesion complexity throughout the cell cycle. Besides RPA, Ataxia telangiectasia and Rad3-related (ATR) was recruited to complex DSBs both in S/G2- and G1-cells. Resection of complex DSBs is driven by meiotic recombination 11 homolog A (MRE11), CTBP-interacting protein (CtIP), and exonuclease 1 (EXO1) but seems not controlled by the Ku heterodimer or by phosphorylation of H2AX. Reduced resection capacity by CtIP depletion increased cell killing and the fraction of unrepaired DSBs after exposure to densely ionizing heavy ions, but not to X-rays. We conclude that in mammalian cells resection is essential for repair of complex DSBs in all phases of the cell-cycle and targeting this process sensitizes mammalian cells to cytotoxic agents inducing clustered breaks, such as in heavy-ion cancer therapy.</p></div

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

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

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

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

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

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