The concept of DNA "repair centers" and the meaning of radiationinduced foci (RIF) in human cells have remained controversial. RIFs are characterized by the local recruitment of DNA damage sensing proteins such as p53 binding protein (53BP1). Here, we provide strong evidence for the existence of repair centers. We used live imaging and mathematical fitting of RIF kinetics to show that RIF induction rate increases with increasing radiation dose, whereas the rate at which RIFs disappear decreases. We show that multiple DNA double-strand breaks (DSBs) 1 to 2 μm apart can rapidly cluster into repair centers. Correcting mathematically for the dose dependence of induction/resolution rates, we observe an absolute RIF yield that is surprisingly much smaller at higher doses: 15 RIF∕Gy after 2 Gy exposure compared to approximately 64 RIF∕Gy after 0.1 Gy. Cumulative RIF counts from time lapse of 53BP1-GFP in human breast cells confirmed these results. The standard model currently in use applies a linear scale, extrapolating cancer risk from high doses to low doses of ionizing radiation. However, our discovery of DSB clustering over such large distances casts considerable doubts on the general assumption that risk to ionizing radiation is proportional to dose, and instead provides a mechanism that could more accurately address risk dose dependency of ionizing radiation. DNA damage-sensing proteins localize at sites of DNA double-strand breaks (DSBs) within seconds to minutes following ionizing radiation (IR) exposure, resulting in the formation of immunofluorescently stainable nuclear domains referred to as radiation-induced foci (RIF) (1-3). RIF numbers are routinely used to assess the amount of DNA damage and repair kinetics after different treatments (4). However, there is a controversy surrounding the question of whether there is a 1∶1 correspondence between RIF and DSBs. For example, pulse field gel electrophoresis (PFGE) analysis suggests that DSBs decay exponentially with time immediately after exposure (5). In contrast, DNA damage-sensing proteins do not instantaneously detect DSBs, leading to delayed kinetics for both detection and resolution. More specifically, the maximum number of 53BP1 or γH2AX RIF is not reached until 15 to 30 min after exposure, and the yield of DSBs predicted by RIF is typically lower than the expected 25-40 DSB∕Gy measured by PFGE (4).Dose response provides another assay for assessing the relationship between DSBs and RIF. Based on theoretical Monte Carlo simulations and PFGE measurements (6, 7), the frequency of DSBs should be highly correlated with radiation dose. Confirming this prediction, two research groups reported that RIF number is proportional to radiation dosage from 1 mGy to 2 Gy (8, 9). In both studies, methods were applied to identify "real" RIF at low doses, where frequencies may be close to background levels before IR (e.g., 10 mGy would lead to about 0.3 DSB∕cell). They either used cells with very low γH2AX background foci (i.e., 0.05 background foci∕cell in primary human l...

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To study the influence of colloidal stability on protein corona formation, gold nanoparticles are synthesized with five distinct surface modifications: coating with citric acid, bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt, thiol-terminated methoxy-polyethylene glycol, dodecylaminegrafted poly(isobutylene-alt-maleic anhydride), and dodecylamine-grafted poly(isobutylene-alt-maleic anhydride) conjugated with polyethylene glycol. The nanoparticles are incubated with serum or bronchoalveolar lavage fluid from C57BL/6 mice (15 min or 24 h) to assess the effect of differential nanoparticle surface presentation on protein corona formation in the air-blood barrier exposure pathway. Proteomic quantification and nanoparticle size measurements are used to assess protein corona formation. We show that surface modification has a clear effect on the size and the composition of the protein corona that is related to the colloidal stability of the studied nanoparticles. Additionally, differences in the composition and size of the protein corona are shown between biological media and duration of exposure, indicating evolution of the corona through this exposure pathway. Consequently, a major determinant of protein corona formation is the colloidal stability of nanoparticles in biological media and chemical or environmental modification of the nanoparticles alters the surface presentation of the functional epitope in vivo. Therefore, the colloidal stability of nanoparticles has a decisive influence on nano-bio interactions.

The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.