Correspondence to J. Lukas: jil{at}cancer.dk
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Abbreviations used in this paper: ATM, ataxia-telangiectasia mutated; DSB, DNA double strand break; H3-dmK79, dimethylated lysine 79 of histone H3; IR, ionizing radiation; MRN, Mre11Rad50Nbs1; siRNA, short interfering RNA.
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Introduction |
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A group of proteins named "checkpoint mediators" play a key role in supporting the timely and effective ATM signaling (Lukas et al., 2004b). Among the checkpoint mediators, 53BP1 has recently attracted particular attention (Mochan et al., 2004). Identified originally as a p53-binding protein (Iwabuchi et al., 1994), 53BP1 was later shown to localize to the DSB sites in cells exposed to ionizing radiation (IR) or radiomimetic drugs (Schultz et al., 2000; Anderson et al., 2001). Indeed, several ensuing observations strongly supported a close functional link between 53BP1 and the ATM-regulated events. First, 53BP1 itself becomes phosphorylated by ATM in a DNA damagedependent manner, suggesting that 53BP1 participates in propagating the ATM signaling to its downstream effectors (Anderson et al., 2001; Ward et al., 2003b). Second, phosphorylation of some ATM targets in 53BP1-deficient mice and human cells is impaired (DiTullio et al., 2002; Wang et al., 2002; Ward et al., 2003a). Third, it has been suggested that 53BP1 may regulate ATM activity by itself (Mochan et al., 2003). Together with the fact that 53BP1 knockout mice suffer from similar (although generally milder) defects as the ATM-deficient mice (Morales et al., 2003; Ward et al., 2003b), the aforementioned findings illustrate that 53BP1 plays an important role in regulating the effectiveness of the ATM-controlled events.
Interestingly, the interaction of 53BP1 with DSBs proceeds in a complex, bimodal fashion. Thus, the assembly at the acute DSB lesions requires direct interaction between the Tudor domain of 53BP1 and dimethylated lysine 79 of histone H3 (H3-dmK79; Huyen et al., 2004). Because this chromatin modification exists in undamaged cells and does not increase in response to DNA damage, it was proposed that chromosomal restructuring adjacent to the DSB lesions locally "unmasks" the methylated lysine residues, thereby allowing their recognition by 53BP1 (Huyen et al., 2004). After establishing the primary contact with DSBs, the retention of 53BP1 in these regions requires another chromatin modification, the ATM-mediated phosphorylation of histone H2AX on serine 139 (-H2AX; Fernandez-Capetillo et al., 2002; Celeste et al., 2003). Unlike H3-dmK79,
-H2AX is low in undamaged nuclei and becomes rapidly induced by DSB-generating insults in chromatin areas flanking each DSB (Rogakou et al., 1999).
These findings raise important conceptual questions: What is the functional interplay between the H3-dmK79mediated assembly and -H2AXdependent retention of 53BP1 at the DSB sites? Are these two phases of DSB53BP1 interaction temporally separated and differently regulated? If so, what is the nature of the molecular switch between them? Furthermore, although H2AX phosphorylation occurs rapidly after DNA damage, how does it becomes relevant for 53BP1 interaction with the DSB regions only later during the DSB response?
To understand the mechanisms (and indeed the purpose) of 53BP1 redistribution after DNA damage, it is important to realize that mammalian cells possess several checkpoint mediators and that all these proteins avidly accumulate in the so-called IR-induced foci. On the one hand, this raises several additional spatiotemporal "problems" such as: How do all these large proteins organize themselves in relatively small areas containing DSBs and limited regions of modified chromatin? Is there a strict "timetable" for an orderly assembly and disassembly of individual checkpoint mediators or do they interact with the DSB microcompartments in a more dynamic and competitive fashion? On the other hand, it is possible that at least some checkpoint mediators may influence each other in terms of their interaction with DSBs and the surrounding chromatin, a notion that might be instrumental to understanding the mechanisms behind early assembly versus late retention of these proteins at the sites of DNA damage. Translated to 53BP1, several papers studied the dependency of 53BP1 accumulation in IR-induced foci as a function of Mdc1/NFBD1 (henceforth Mdc1), another recently characterized checkpoint mediator (Stucki and Jackson, 2004). Although intriguing, these studies reached conflicting results and left a considerable confusion regarding the requirement of Mdc1 for 53BP1 interaction with the DSB sites (Mochan et al., 2004). Thus, whereas two studies reported productive formation of 53BP1-decorated foci (Goldberg et al., 2003; Mochan et al., 2003), another study failed to detect any focal accumulation of 53BP1 in Mdc1-deficient cells after IR (Stewart et al., 2003).
Resolving these issues appears important for several reasons. Most notably, 53BP1 appears to be the closest structural and functional homologue of the "ancestral" checkpoint mediator in unicellular organisms (Rad9 in Saccharomyces cerevisiae and Crb2 in Schizosaccharomyces pombe, respectively; Mochan et al., 2004). In this sense, deeper insight into its nuclear dynamics may not only clarify the lingering discrepancies about 53BP1 itself but it can also elucidate some of the outlined conceptual questions of how checkpoint mediators interact with DSBs. In the present study, we performed a detailed spatiotemporal analysis of intranuclear redistribution of 53BP1 in its physiological environment: the nucleus of a living mammalian cell. Our results provide new insights into cooperation between Mdc1 and 53BP1 checkpoint mediators in organizing the chromatin microenvironment around the DSBs, help explain the hitherto elusive molecular switch between the assembly and retention of 53BP1 at the DSB sites, and provide evidence for a key role of Mdc1 in mediating sustained interaction of DSB regulators with the DNA damagemodified chromatin.
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Results |
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The Tudor domain determines intranuclear mobility of 53BP1 both before and after DNA damage
Next, we exploited the recent discovery that the Tudor domain of 53BP1 directly interacts with H3-dmK79 (Huyen et al., 2004), thereby determining the productive accumulation of 53BP1 in the IR-induced foci. We have generated another U-2-OSderived cell line stably expressing GFP-tagged murine 53BP1 with an inactivating mutation (Y1487L) in the core part of the Tudor domain. As predicted, this Tudor-deficient 53BP1 completely failed to assemble along the laser-generated DSB tracks (Fig. 2 D). Surprisingly, FRAP measurements revealed that the Tudor domain disruption accelerated the 53BP1's mobility also in undamaged nuclei manifested by a faster recovery of the GFP-53BP1-Y1487L and a significant decrease of its intranuclear residence time (Fig. 2 E and Table I). Thus, the integrity of the Tudor domain appears to have an impact not only on the ability of 53BP1 to assemble around the DSB lesions but also on its physiological rate of binding to chromatin during unperturbed cell cycle progression (see Discussion).
The assembly of 53BP1 at the DSB sites lags behind that of Mdc1
To gain deeper insight into the dynamics of 53BP1 interaction with DSB-surrounding chromatin, we cross-examined redistribution of 53BP1 and Mdc1 in living cells exposed to the laser-generated DSB tracks. This strategy was inspired by our findings that the assembly of Mdc1 at the DSB sites is required to transiently immobilize the Mre11Rad50Nbs1 (MRN) complex (DSB sensor and a key component of early ATM-controlled signaling) in these regions (Lukas et al., 2004a). Given this capacity of Mdc1 to "organize" other proteins within the DSB-flanking chromatin, we tested whether it also contributes to 53BP1 assembly at the DSB sites on a complete checkpoint time scale.
First, we compared the kinetics of 53BP1 and Mdc1 assembly at the freshly generated DSBs. To ensure identical experimental conditions, we cocultivated the GFP-53BP1expressing M1 cells with U-2-OS cells stably expressing GFP-Mdc1 (Lukas et al., 2004a). To discriminate these two cell lines during imaging (both contain GFP-tagged proteins), we labeled the GFP-Mdc1expressing cells with a cytosolic cell tracker detectable within the red ( = 600 nm) emission spectrum (Fig. 3 A; and see Materials and methods). Real-time imaging of these mixed cultures exposed to laser microirradiation revealed that the assembly of GFP-53BP1 in the DSB areas was delayed and proceeded with a significantly slower kinetics compared with that of GFP-Mdc1 (Fig. 3, A and B; and compare
values in Table I). This temporal difference in Mdc1 and 53BP1 accumulation at the DSB sites was confirmed also for the endogenous proteins analyzed by immunofluorescence in the same cells fixed in short intervals after laser microirradiation (Fig. 3 C). Together, these data suggest that the DSB-associated local chromatin modifications that direct the respective checkpoint mediators to the sites of DNA damage are generated (or become accessible) with different kinetics.
Mdc1 is required for a productive assembly of 53BP1 at the DSB sites
The aforementioned observation prompted us to investigate whether Mdc1 (the faster DSB interactor) influences the kinetics and/or the magnitude of 53BP1 assembly at the DSB sites. To this end, we quantitatively depleted endogenous Mdc1 in M1 cells by short interfering RNA (siRNA; Fig. 4 A, left) and measured the increase of the GFP-53BP1associated fluorescence after laser microirradiation. Strikingly, these analyses showed that the saturation amounts of GFP-53BP1 in the microirradiated regions were drastically reduced compared with the Mdc1-proficient cells (Fig. 4 A, right; Fig. 4 B; and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200503043/DC1). Similar impairment in 53BP1 assembly at the DSB sites was observed also in Mdc1-deficient cells exposed to IR (Fig. S1 B). In a reciprocal experiment, an equally quantitative, siRNA-mediated depletion of 53BP1 from the GFP-Mdc1 cells (Fig. 4 C, left) had no significant effect on the kinetics of GFP-Mdc1 assembly around the laser-generated DSBs (Fig. 4 C, right). Together with the aforementioned temporal distinctions between the Mdc1 and 53BP1 assembly kinetics (Fig. 3), and additional FRAP measurements revealing that the Mdc1 depletion reduced the size of the slow binding fraction of GFP-53BP1 in the microirradiated areas (Fig. S2 B), these data suggest that the lack of Mdc1 causes severe limitations for 53BP1 to interact with the DSB sites, including the earliest stages of the DSB response.
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Discussion |
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In addition to integrating 53BP1 to the spatiotemporal "map" of the DSB response, interesting conclusions could be derived from the analysis of its mobility in undamaged nuclei. The distribution of the nuclear pool of 53BP1 into fast and slow moving fractions (Table I) indicate that even in the absence of DNA damage 53BP1 associates with chromatin (Phair et al., 2004). This basal 53BP1chromatin interaction is transient (thereby allowing relatively rapid movement and availability of a sizable pool of 53BP1 throughout the nucleus), it is partly dependent on the integrity of the methyl-binding Tudor domain (suggesting that H3-dmK79 could be transiently exposed during unperturbed cell cycle, likely as a result of the dynamic conformational changes of nucleosomes; Li et al., 2005), and could be instantly converted to a more sustained immobilization of 53BP1 at the acutely emerging sites of DNA breaks. Such protein redistribution after DNA damage is not unprecedented. Rapid movement of repair factors and their transient immobilization after stochastic collisions with DNA lesions was originally described for proteins involved in the nucleotide excision repair (Houtsmuller et al., 1999) and later confirmed as a prevailing way of protein communication with other types of DNA lesions including DSBs (Essers et al., 2002; Lukas et al., 2003, 2004a).
How does Mdc1 influence 53BP1 redistribution? One parameter that can explain the gain of Mdc1's ability to organize 53BP1 and other proteins specifically in the context of DNA damage is its own, ATM-mediated phosphorylation (Stucki and Jackson, 2004). This idea was originally inspired by our finding that the switch between transient interaction and sustained accumulation of MRN at the DSB sites requires both Mdc1 and the intact FHA domain of Nbs1 (Lukas et al., 2004a). As the FHA domains bind DNA damage-induced phosphothreonines (Durocher et al., 2000), it is likely that both physical presence and phosphorylation of Mdc1 determine the increased immobilization of Nbs1 in the DSB regions (Fig. 7). Although the 53BP1 contains a tandem BRCT domain, which is an equally avid phosphothreonine interactor (Manke et al., 2003; Yu et al., 2003), its accumulation at the DSB sites seems to differ from the Mdc1-Nbs1 interplay, because deletion and/or mutation of the BRCT domains neither changed the assembly nor modified the residence time of 53BP1 at the DSB sites (unpublished data). Instead, we propose that after its assembly around DSBs and phosphorylation by ATM, Mdc1 gains the ability to facilitate the recruitment of chromatin-remodelling enzymes (Fig. 7), such as those that had been reported to interact with phosphorylated histone H2A in yeast (Downs et al., 2004; Morrison et al., 2004; van Attikum et al., 2004). The ensuing nucleosomal rearrangements may then promote and/or stabilize exposure of H3-dmK79. An alternative (but not mutually exclusive) scenario is that the presence of phosphorylated Mdc1 at the DSB-flanking chromatin directly stabilizes binding between 53BP1 and interaction-competent H3-dmK79 residues. Thus, together with the recent biochemical evidence for its ability to integrate multiple DSB regulators at the sites of DNA damage (Goldberg et al., 2003; Lou et al., 2003b; Peng and Chen, 2003; Stewart et al., 2003; Xu and Stern, 2003), Mdc1 appears to perform a key role in organizing the DSB-surrounding chromosomal microenvironment, a function that may have important biological ramifications. In particular, the inability to concentrate proteins such as MRN and 53BP1 at the DSB sites in Mdc1-deficient cells could impair timely and productive DSB repair and thereby explain the decreased survival of such cells reported previously in clonogenic assays (Lou et al., 2003a; Stewart et al., 2003) and confirmed in this study by the real-time imaging of individual cells (Table II).
Finally, the quantitative and temporal aspects of our measurements, combined with a direct comparison of intranuclear protein redistribution under identical experimental conditions, challenge another intensively debated issue, namely the nature of the mechanisms that sense DSB lesions and initiate the genome surveillance program. In particular, it has been proposed that Mdc1 and 53BP1 constitute two branches of cellular mechanism to activate ATM-dependent signaling in cells exposed to DSBs (Mochan et al., 2003). However, our new data showing that the assembly of 53BP1 at the freshly generated DNA lesions lags behind Mdc1 and that the proficient 53BP1-DSB assembly is indeed dependent on Mdc1 are not consistent with 53BP1 being the prime sensor of DSBs. Moreover, our previous results strongly suggest that Mdc1 is not involved in the initial DSB detection either. The key arguments here are that Mdc1 is not a constitutive structural component of the MRN complex (the likely DSB sensor; see the following paragraph) and that the interaction of Mdc1 with the DSB-flanking chromatin is critically dependent on the H2AX phosphorylation (i.e., downstream of at least the first "wave" of ATM activity; Lukas et al., 2004a). Instead, our data indicate that both Mdc1 and 53BP1 function as genuine "mediator" and/or "adaptor" proteins and that their main role is to enhance and locally concentrate the efficiency of multiple interactions between activated ATM and its substrates, thereby facilitating the pace and velocity of the ATM-controlled signaling.
What, then, is the true DSB sensor? Kinetic measurements in yeast (Lisby et al., 2004) and our own results in mammalian cells (Lukas et al., 2004a) revealed that so far the only nuclear factor whose productive interaction with DSBs does not seem to require other proteins and/or protein modifications and whose arrival at the freshly generated DSBs precedes that of all DSB regulators analyzed so far is the MRN (MRX in S. cerevisiae) nuclease complex. Unlike 53BP1, the MRN complex has a distinctly dual mode of interaction with the DSB sites, including direct and transient binding to DNA (D'Amours and Jackson, 2002) and the more sustained, chromatin-dependent accumulation in the DSB-flanking chromatin (Lukas et al., 2004a). Although the first mode (Mdc1 independent) is compatible with the proposed role of MRN as a DSB sensor, the second one (Mdc1 dependent) likely serves other purposes such as preventing dispersal of the ATM-activated MRN to the undamaged nuclear compartments (Lukas et al., 2004a). In contrast, the recent biochemical analyses (Huyen et al., 2004) revealed no evidence for the direct interaction of 53BP1 with DNA, a notion fully consistent with our present single cellbased approach. Thus, unlike the MRN complex (that can interact with DSBs in both DNA-dependent and chromatin-dependent modes), the productive assembly of 53BP1 at DSBs relies on a single interaction mode that is largely (if not exclusively) dependent on temporally preceding chromatin modifications, specifically on the formation of the -H2AXMdc1 complex. Together, this strongly suggests that although 53BP1 may reinforce DSB signaling, it is not the primary DSB sensor.
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Materials and methods |
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Cell culture and RNA interference
For generation of stable cell lines, the human U-2-OS osteosarcoma cells (American Type Culture Collection) were cotransfected with the expression plasmids containing various forms of GFP-53BP1 and the pBabe-puro containing the puromycin resistance cassette. Upon selection with 1 µg/ml puromycin (Sigma-Aldrich) for 10 d, resistant clones were tested for the expression and functionality of the GFP-tagged proteins. The U-2-OSderived cell line stably expressing GFP-Mdc1 was described previously (Lukas et al., 2004a). For live-cell experiments, the cells were plated on the Lab-Tek chambered coverglass (Nunc) or on Cellocate grid coverslips (Eppendorf). The culture medium was supplied with 10 µM BrdU (Sigma-Aldrich) for 24 h to sensitize the cells for DSB generation by UV-A laser (Lukas et al., 2003, 2004a). For every live-cell manipulation and/or recording, the cells were supplied with a phenol red-free, CO2-independent medium (Invitrogen). For distinction of GFP-53BP1 and GFP-Mdc1expressing cells during simultaneous imaging of mixed cell cultures, the GFP-Mdc1 cell line was labeled with the red-emitting CMTPX Cell Tracker (Molecular Probes) according to the manufacturer's instructions. IR was delivered by X-ray generator (Pantak HF160, 150kV, 15mA, dose rate 2.18 Gy/min) as described previously (Syljuasen et al., 2004). The Mdc1-targeting siRNA duplexes were described previously (Lukas et al., 2004a). The 53BP1-targeting siRNAs were designed and used as described in DiTullio et al. (2002). The ATM-targeting siRNA was a Smartpool (Dharmacon). Control siRNA (5'-gggaggacaagacguucua-3') was against HSP70B (Leung et al., 1990), a variant of the human heat shock protein that is not expressed in U-2-OS cells. All siRNAs were synthesized by Dharmacon research.
Antibodies and immunochemical techniques
Mouse mAbs against 53BP1 and ATM (MAT3) ware gifts from T. Halazonetis (Wistar Institute, Philadelphia, PA; Schultz et al., 2000) and Y. Shiloh (Tel Aviv University, Tel Aviv, Israel), respectively. Additional antibodies used in this study included: rabbit anti-Smc1 (Abcam), rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), rabbit anti-53BP1 (Oncogene Research Products), rabbit 53BP1 (Santa Cruz Biotechnology, Inc.), rabbit and mouse anti-H2AX (Upstate Biotechnology). Rabbit antibody to Mdc1 was provided by S. Jackson and M. Stucki (The Wellcome Trust/Cancer Research Institute, Cambridge, UK; Goldberg et al., 2003). Highly cross-adsorbed secondary antibodies for immunofluorescence coupled to Alexa 488 or Alexa 568 were purchased from Molecular Probes. Conditions for immunostaining, SDS-PAGE electrophoresis, and immunoblotting were described previously (Falck et al., 2002; Lukas et al., 2003, 2004a).
Microscopy
Confocal microscopy of fixed cells was performed on LSM 510 (Carl Zeiss MicroImaging, Inc.) mounted on a microscope (model Zeiss-Axiovert 100M; Carl Zeiss MicroImaging, Inc.), equipped with Plan-Neofluar 40x/1.3 oil immersion objective, and with appropriate configurations for multiple color acquisition. For live cell confocal microscopy, a custom-designed imaging workstation combining the LSM 510 Meta (Carl Zeiss MicroImaging, Inc.) and the P.A.L.M. microdissector (PALM Robotics) was used. A 40x/1.2 C-Apochromat water immersion objective was used both for microirradiation and image acquisition. Generation of local DSB regions by laser microirradiation and the FRAP assays were described previously (Lukas et al., 2003, 2004a). Given these objective parameters the energy output of the microdissection laser that is compatible with generating local DSB tracks ranges between 51, 55, and 59%, yielding low, intermediate, or high concentration of local DSBs (Lukas et al., 2003). Unless stated otherwise, the 55% laser energy output was used. Time-lapse experiments were performed on a widefield fluorescence microscope (Axiovert 200; Carl Zeiss MicroImaging, Inc.) equipped with Plan Neofluar 25x/0.8 oil immersion objective, a charge-coupled device camera (Coolsnap HQ; Roper Scientific), and Metamorph software (Universal Imaging Corp.). A typical time-lapse acquisition protocol consisted of a 5-min interval autofocus on a differential interference contrast transmission light image (10-ms exposure time) followed by one snapshot of a GFP image with an exposure time of 100 ms. All microscopes used for live cell imaging were equipped with a 37°C tempered XL incubator and the culture medium was overlaid with mineral oil to prevent evaporation. For quantitative and comparative imaging, identical image acquisition parameters were used. Software packages used to capture the images, analyze the data, and generate the graphs included LSM (Carl Zeiss MicroImaging, Inc.), Metamorph (Universal Imaging Corp.), Excel (Microsoft), and Prism 4 (Graphpad Software).
Real-time assembly assays
Operation of the combined confocal and microdissection microscope was performed essentially as described previously (Lukas et al., 2003, 2004a), with the addition that time-lapse series for dynamic measurements of DSB-induced protein redistribution were acquired as sets of 50 pictures averaged four times (acquisition time = 4 s) with intervals of 15 s between individual frames. Fluorescence redistribution data from regions encompassing the irradiated tracks of at least 10 cells per experiment were extracted from these time-lapses. The fluorescence values of individual frames were annotated so that It denotes the intensity of the measured region at time point t, whereas Io and I denote the values measured in the first and last frame, respectively. Additionally, three values for each measured cell were recorded: (1) background fluorescence outside the nucleus (Ibg), (2) basal intensity within the nucleus in the first frame (Ipre), and (3) the peak intensity within the DSB track in the last frame (Iend). These data were combined in two different ways to normalize the fluorescence output from individual cells. In the first way, post-normalization of fluorescent units (Post NFU) translates the data into a kinetic profile of numbers between 0 and 1. These numbers were used for mathematical modelling in the following equation: Post NFU = (It Io)/(I
Io). The second way was fold increase normalization, which is an absolute measure for the increase in fluorescence within the DSB-containing nuclear regions over time. In this case, the mathematical evaluation of the data was done as follows: Fold increase = 1 + [Post NFU x (Iend Ipre)/(Ipre Ibg)].
For kinetic modelling, post value normalized data were fitted to solutions of linear differential equations of increasing order. Thus, the two following one-parameter first and second order, respectively, kinetic models for the redistribution profiles were considered:
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Application of the second order model gave the best fit using the Prism 4 software. Therefore, the parameter was used to compare the protein's assembly kinetics for various conditions specified in the figure legends. The same mathematical procedure was applied to model real-time assembly of Mdc1 (Fig. S2 A). In this case, both the first and second order model fitted well the experimental data (Lukas et al., 2004a), and the second order model was used here to directly compare the kinetic assembly of Mdc1 with that of 53BP1 (the latter could be satisfactorily described only by the second order model [Fig. 2 B]).
Photobleaching protocols
The nuclei of M1 cells were microirradiated, and the GFP-53BP1 protein was allowed to reach its maximal steady-state concentration in the DSB-containing nuclear tracks (typically 20 min after laser exposure). Subsequently, a narrow, 2-µm-wide rectangular region was placed over the entire DSB track and/or the undamaged nucleoplasm as indicated in the figure legends. After acquisition of five prebleach images, this region was subjected to a single bleach pulse of five iterations at a laser transmission of 100%. Subsequently, 95 images were acquired in 1-s intervals with 0.5% laser transmission. Averaged fluorescence intensities of the whole or parts of these regions were extracted and normalized either to the post-bleach value (see Post NFU equation in previous section) or to the prebleach value as follows: Pre NFU = (It Io)/(Ipre Io). In the latter case, Io stands for the intensity in the first frame after the bleach pulse, I for the average of the five last measurements, and Ipre for the average of the five prebleach measurements. Post-normalized fluorescence (see Post NFU equation in previous section) values from the strip FRAP experiments were used for kinetic modelling. Assuming two populations of binding reactions between mobile 53BP1 and immobile chromatin, the relative abundance (Y) of the 53BP1 populations engaging in the different binding reactions and the time spent within the bleach region (
) of protein molecules from each population were modeled for different proteins and conditions according to the following equation (Schmiedeberg et al., 2004):
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Online supplemental material
Fig. S1 shows that the timing of GFP-53BP1 assembly in the IR-induced foci recapitulates that of the endogenous protein and that Mdc1 ablation by siRNA attenuates formation of the GFP-53BP1 foci. Fig. S2 shows the kinetic modeling of GFP-Mdc1 assembly at the microlaser-generated DSB sites and the impaired binding of GFP-53BP1 to the DSB-flanking chromatin in Mdc1-depleted cells assayed by FRAP. Fig. S3 shows the inefficient assembly of GFP-53BP1 in a microirradiated Mdc1-deficient cell when compared with an Mdc1-proficient cell recorded in the same microscopic field. Fig. S4 shows that the lack of Mdc1 triggers premature dissociation of GFP-53BP1 from the sites of extensive DNA damage generated by high-energy laser irradiation. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200503043/DC1.
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Acknowledgments |
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This work was supported by grants from the Danish Cancer Society (DP 03 035), Danish National Research Foundation, European Union (DNA Repair 512113), European Science Foundation (EuroDYNA 21-04-280), and John and Birthe Meyer Foundation.
Submitted: 8 March 2005
Accepted: 6 June 2005
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