Institute of Molecular Biotechnology Jena, Beutenbergstr. 11, 07745 Jena, Germany
* Author for correspondence (e-mail: bar{at}imb-jena.de)
Accepted 10 June 2004
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Summary |
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Key words: DNA double strand break, Gamma-H2AX, Homologous recombination repair, Nonhomologous end joining, Rad51 foci, Ultraviolet radiation
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Introduction |
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The Rad50/Mre11/NBS complex serves as an initial factor and has been reported to participate both in the NHEJ and in the HRR pathway (Jackson, 2002). In the NHEJ, the complex is assumed to modify the ends of the DSB site by its endo- and exo-nuclease activity (Trujillo et al., 1998
). In HRR, the complex acts as an exonuclease to produce single-strand overhangs (Jackson, 2002
). The central proteins of the NHEJ pathway are the Ku70/80-heterodimer and DNA-PC (Allen et al., 2002
), which rejoin the broken ends. Finally the Ku protein recruits the XRCC4, a cofactor of Ligase IV, which is a phosphorylation target of DNA-PK (Leber et al., 1998
) that, in turn, enhances the recruitment process to close the strand break.
HRR is also initiated by the NBS1/Mre11/Rad50 complex that incises the two strands and produces 3' overhangs that are bound by Rad52. The single-stranded DNA is bound by multiple Rad51 molecules (Haaf et al., 1995; Raderschall et al., 2002
). Strand invasion and recombination are enhanced by Rad51, its paralogues and Rad54 (Hoeijmakers, 2001
; Valerie and Povirk, 2003
) and finally ligated by ligase I.
Presently it is assumed that in mammalian cells the NHEJ system is the predominant one, whereas the HRR is active in late S and G2 only. While this relationship has been recently studied in hamster cells (Rothkamm et al., 2003) the data for human cell systems are more controversial (Valerie and Povirk, 2003
). Also, it is not defined to which degree the two repair pathways contribute to the overall DSB repair and to DSB repair in specific cell-cycle phases.
For a more-detailed insight into DSB repair and NHEJ/HRR crosstalk, knowledge on network-like interactions in spatial and temporal terms is required. We have measured the interaction in the repair-proficient human cell line, HaCaT, with eight proteins from the two pathways. The interaction matrix of several pairs of repair protein allows us to propose that the co-activation of both DSB repair pathways on the same DSB is a general response of the cell in the S or G2 phases, if both systems are available.
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Materials and Methods |
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Comet assay
Comet assay was performed according to the alkaline version described by Singh and Tice (Bauer et al., 1998; Singh et al., 1988
; Tice et al., 2000
). In short, the incubation times were as follows: 1 hour lysis at 4°C, 1 hour unwinding at 4°C and 30 minutes electrophoresis at 1 V/cm also at 4°C. The neutral Comet assay was performed according to the procedure described elsewhere (Olive and Banath, 1993
), with an additional proteinase K digest for 4 hours at 37°C. Also, electrophoresis was performed at 4°C in 1x TBE at 1 V/cm for 30 minutes. Image acquisition and analysis was performed with the KOMET 4© system (Kineticimaging, UK). 4x2 slides with 60 Comets each of individual preparations were scored per sample point.
Micronucleus assay
After irradiation, cells grown on cover slips were incubated for 24 hours in the presence of cytochalasin B to block cell, but not nuclear, division at a final concentration of 2 µg/ml in RPMI medium to identify binucleated cells. After fixation, DNA was stained with Hoechst dye and the cytoskeleton (to identify the cytoplasm) was labelled with phalloidin-rhodamin diluted 1:1000 in PBS for 30 minutes. Specimens were analysed by combined fluorescence and phase contrast microscopy. 4x500 binucleated cells were analysed per sample point of two individual preparations.
Immunohistochemistry
Cells were grown on microscopic glass slides and washed once in PBS. Fixation was done with 3.7% formaldehyde and permeabilization with 0.7% Triton X-100 using standard conditions. After blocking with 3% BSA in PBS for 1 hour, primary antibodies (rabbit anti-Rad51: H-92, goat anti-Rad51:C-20, both from Santa Cruz Biotech, USA; rabbit anti -H2AX, Travigen, USA; mouse anti
-H2AX, Upstate, USA; mouse anti-DNA-PKcs Kamiya, USA; rabbit anti-Rad50 H-300, goat anti-Mre11 C-16, rabbit anti-Rad52; goat anti-XRCC4, all from Santa Cruz Biotech, USA; mouse anti-Ku70, Sigma-Aldrich, Germany and mouse anti-Mre11, R&D Systems, USA) were added, at a dilution of 1:200 in PBS containing 2% BSA and incubated for 1 hour at room temperature. For single staining secondary antibodies [anti-rabbit FITC, anti-mouse-FITC or anti-goat-FITC (all Sigma Aldrich), diluted 1:200 in PBS containing 2% BSA] were used for the detection. For two colour immunohistochemistry donkey anti-goat, donkey anti-rabbit and donkey anti-mouse conjugated with either Alexa488 or Alexa594 (all from Molecular Probes) were used at a dilution of 1:400 and were applied as described above.
Microscopy and imaging
Microscopic imaging was done using an Axioskop (Zeiss, Jena) equipped with a cooled CCD camera (Photometrix, Quantix) and a HBO50 illumination. High numerical objectives were used together with immersion oil: 40x NA1.3; 63x NA1.25; 100x NA1.3 (Planneofluar series, Zeiss, Germany). The camera was controlled by the Qips software package (Vysis, Germany) and by Openlab (Improvision, UK). For fluorescence excitation/emission, high-quality band-pass filters (Chroma, USA) were used. Images were captured as 12 bit black and white images and stored in TIF format. Contrast and brightness were adjusted using Adobe Photoshop before images were merged as pseudo-colour. Confocal imaging was done using a LSM 510 (Zeiss, Jena) equipped with an Ar ion and an HeNe laser, controlled by the LSM 3.0 software (Zeiss, Germany). Single images were scanned 4x per pixel and a median filter was applied during acquisition to reduce noise and then exported as TIF images for each channel individually, and processed in Photoshop as described above. FRET imaging was done using the LSM 510 by exciting the Alexa488 (donor) dyes at 458 nm to avoid direct excitation of the acceptor dye (Alexa594). Emission of the acceptor was detected by a LP 580 nm filter. For representation the images were binarised to black and white and exported in single TIF files.
Western blotting
Western blotting was performed after total protein isolation with RIPA buffer (1x PBS, 1% Nonidet P-40 0.5% sodium deoxycholate, 0.25% SDS, 100 µg/ml PMSF, 1 tablet of protease inhibitors (Complete Mini, Roche) following standard procedures. 50 µg of total protein were separated on a 10% PAA gel and transferred to a nirocellulose membrane. The same primary antibodies as mentioned above were used at a dilution of 1:1000 in PBS including 4% BSA and 0.05% Tween 20. Secondary antibodies (HRP conjugated) were used together with the ECL Plus chemoluminiscent detection kit (Amersham Pharmacia).
Immunoprecipitation (IP)
For IP 2x107 cells were chemically cross-linked with 2.8% formaldehyde in PBS for 15 minutes at room temperature. After the reaction the formaldehyde was quenched with 1/15 volume of 2 M glycine for 5 minutes at room temperature. Cells were harvested by centrifugation, washed in cold PBS and resuspended in ice-cold IP-buffer (10 mM Tris HCl pH 8.0, 140 mM NaCl, 0.025% NaN3, 1% Triton, 0.05% SDS, 1% sodium deoxycholate) including protease inhibitors and PMSF, and were lysed for 30 minutes on ice. DNA was fragmented by three cycles of sonication at 60% output for 10 seconds. After 2x10 minutes centrifugation at 10,000 g at 4°C the supernatant was transferred to a fresh tube and pre-cleared with 15 µl/ml anti-goat-agarose (respective anti-rabbit- or anti-mouseagarose, all from Sigma-Aldrich, Germany) for 1 hour at 4°C. The agarose beads were separated by centrifugation and the supernatant was transferred to a fresh tube. 10 µl of the primary antibody were added per 500 µg of pre-cleared protein extract and incubated over night at 4°C. The protein complex was then precipitated after 1 hour of incubation with the antibody-coupled agarose at 4°C by centrifugation. The pellet was washed 3x with 0.2 ml IP-buffer. The agarose pellet was resuspended in 50 µl 1xSDS-loading buffer (45 mM Tris HCl pH 6.8, 10% Glycerol, 0.5% SDS, 0.01% Bromphenol Blue, 0.05 M DTT) and loaded on a 10% SDS gel after boiling for 5 minutes. The detection was performed after blotting as described above.
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Results |
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DNA single-strand breaks are induced 3.5-fold and repaired more than six times faster than double-strand breaks
Induction and repair of DNA single- and double-strand breaks as measured by the Comet assay (Fig. 1) together with sample micrographs of typical Comets (Fig. 1D-E). DNA single-strand breaks together with alkali-labile sites, such as abasic sites, are induced in a dose-dependent (i.e. fluence-dependent) manner as measured with the alkaline version of the Comet assay. The DNA damages increase linearly with exposure time and with fluence. Strictly this increase should follow an exponential function, such as described in Eqn 2 (below). Because obviously DNA damage is far from saturation, a linear approximation is adequate. After irradiation, the DNA damages recover following an exponential decay (Fig. 2B) (in particular, see inset Fig. 2B).
![]() | (1) |
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The time constant for the single-strand break repair can be calculated as
=110 minutes. From the time constant the time period of the SSBs in the alkaline Comet assay can be calculated to TG=75 minutes.
With the neutral Comet assay, double-strand breaks are detected exclusively. This version of the Comet assay omits the DNA denaturation step and, therefore, only fragments generated by DSBs can migrate in the electric field. As with the induction of single-strand breaks, the induction of DSBs is linearly dependent on the fluence. Repair of the DSBs is found to be slower than repair of the SSBs. The residual level of DNA damage after 5 hours of repair is still significantly higher than the control level (one-sided Anova, Dunnett's method, P<0.05). For the DSBs it takes 24 hours post-irradiation until the level of damaged DNA is reduced to the control level. The initial repair of DSBs follows Eqn 1 with =670 minutes and a half-life period of approximately 460 minutes (Fig. 1B). Although single-strand breaks highly exceed double-strand breaks, the latter are clearly detectable.
The linear dependence of DNA damage induction on the fluence (or exposure time) reveals information about the repair process. Even in a region far from saturation, a decrease in the detected SSBs is expected, because DNA repair should counteract damage induction. This discrepancy, at a first glance, can be explained by the fact that the Comet assay does not only detect radiation-induced DNA damages, but also the nicks and gaps that are generated during base excision repair (BER) and nucleotide excision repair (NER) (Asaeda et al., 1998; Collins and Squires, 1986
; de With and Greulich, 1995
; Kleinau et al., 1997
; Olive and Banath, 1993
). The repair time measured in our experiments is slow compared with other reports in the literature (Hamilton et al., 2001
; Terris et al., 2002
), where half-life times of a few minutes to 30 minutes (for SSBs and base damages) have been reported. A possible reason for this is the high UV-A fluences of 960 kJ/m2 used for the repair study. This high fluence induces a large number of base modifications (mainly 8-oxo-G) and SSBs. The repair of these damages involves NER and BER, two systems that induce further nicks, which are not discriminated by the Comet assay from initial damage. Therefore, it seems reasonable that a prolonged repair time is detected because the removal of SSBs is overlaid by repair processes that induce further nicks.
So far the dose relationship of DNA damages has been described in time constants. The following data will be described in terms of fluence constants, which are related to the time constants via the fluence rate.
Induction of micronuclei
The induction of micronuclei is accepted as a further indication for DNA double-strand and/or chromatin breaks (Kasahara et al., 1992; Stopper et al., 1994
). Micronuclei (MN) are formed during mitosis, when chromatin fragments are not distributed into daughter nuclei. We examined induced micronuclei as a function of irradiation time and fluence (Fig. 2A). The inset of Fig. 2A shows a sample micrograph with a micronucleus (arrow). The MN frequency in control cells is 2.7±0.4% and increases significantly to 5% after a fluence of 320 kJ/m2. After 30 minutes irradiation (corresponding to a fluence of 960 kJ/m2) the MN frequency reaches the saturation value of 10.6±0.5%. The MN frequency does not increase further but remains on the same level after 40 minutes of irradiation. An exponential increase to a maximum can be described according to the following equation (Eqn 2)
![]() | (2) |
where M is the maximum value, with M=12.7. The fluence constant is calculated to F=784.8 kJ/m2 or can also be expressed as time constant , because the fluence is directly proportional to the time.
The obtained values confirm previous data (Phillipson et al., 2002), where comparable values for MN induced by UV-A have been reported and where it was demonstrated that these damages persist up to 21 days post exposure. The flattening of the curve at fluences above 1000 kJ/m2 can be explained with a reduced viability. To verify the induction of DSBs, the formation of
-H2AX foci were monitored by immunofluorescence.
Immunohistochemical detection of DSB sites
The number of cells with -H2AX signal is plotted against the irradiation time (Fig. 2B). Sample micrographs of the detected foci in control cells and after 30 minutes of irradiation are shown in the inset of Fig. 2B. The number of cells with
-H2AX foci increases from 11.1±1.2% (controls) to 43.0±1.7% after only 5 minutes of UV-A exposure (corresponding to 180 kJ/m2). The number of cells with
-H2AX signal reaches 85.2±9.3% after 10 minutes of irradiation. Further exposure does not significantly increase the number of cells with
-H2AX signal (Phillipson et al., 2002
). According to Eqn 2 the induction yields M=93.5 and F=223 kJ/m2. In terms of exposure time,
is found to be 6.2 minutes. The involvement of NHEJ in UV-A-induced DNA double-strand-break repair has recently also be demonstrated (Fell et al., 2002
).
NHEJ activity after UV-A irradiation
NHEJ was monitored by the formation of DNA-PKcs foci. The time-dependent formation of the foci was quantified as cells that exhibit foci after irradiation. Values for the induction, together with two sample micrographs showing non-irradiated cells (left) and after 20 minutes UV-A (right), have been plotted (Fig. 2C). In non-irradiated cells almost no foci are found 1.6±0.4%, but a diffuse labelling instead. After 5 minutes of exposure, 23.4±5.3% of the cells show foci. The number increases further to 64.2±15.3% after 10 minutes and reaches the maximum after 20 minutes of exposure at 84.6±11.9%. Further irradiation does not increase the number of cells with foci. According to Eqn 2, the time constant for the induction of DNA-PKcs foci is 9.1 minutes, which equals a fluence constant of F=290 kJ/m2, with M=85.6.
Activation of homologous recombination repair after UV-A exposure
To follow the repair of DSBs, the HRR activation was studied by the visualisation of Rad51 foci. To quantify this, the number of cells showing Rad51 foci is quantified in unsynchronised cells. The percentage of cells with Rad51 foci, together with two example micrographs showing typical cells before and after the exposure to UV-A, was plotted (Fig. 2D). Rad51 foci formation is thought to represent HRR (Haaf et al., 1995). The initial level of Rad51 foci before irradiation is 2.7±1.9%, increases after 10 minutes to 25.7±5.5% and reaches saturation after 30 minutes at 42.2±2.9% of the cells showing Rad51 foci. The curve can again be described by Eqn 2 with M=42.7, and F=360 kJ/m2 (e.g.
=11.25). For a direct comparison of the induction rates the normalised (to the maximum) values are plotted together (Fig. 2E).
Cell-cycle-dependent activation of repair pathways
The saturation limit of approximately 40% of the cells that show Rad51 foci after UV-A exposure correlates with the potential activation of the HRR pathway during the cell cycle. It has been reported that HRR is especially active in late S and G2 cells, where the homologous sequences have been already replicated (Rothkamm et al., 2003; Hoeijmakers, 2001
). To test this hypothesis we exposed cell-cycle-synchronised cultures to a single fluence of 960 kJ/m2, and analysed the number of cells with Rad51 and
-H2AX foci. We found that in G1 only 7.2±4.1% of the cells show Rad51 DNA-PKcs foci, whereas 86.3±3.8% of the cells were Rad51-positive in cultures after 9 hours following a thymidine block. By contrast, the number of cells with
-H2AX and DNA-PKcs foci is found to be independent of the cell-cycle position, and foci were found in 90.1±5.8% and 80.7±4.2% of the cells, respectively, in all examined cell-cycle phases.
The values for the frequency of Rad51, DNA-PKcs and -H2AX foci in unsynchronised G1- and G2-enriched cultures are shown (Fig. 3A). Sample images (Fig. 3B-D) and the cell-cycle distribution of the cultures after DNA staining and flow cytometry analysis (Fig. 3E).
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These results, together with the finding that the number of Rad51-positive cells is dependent on the cell cycle rather than on the UV-A fluence, explain the saturation at 40% of cells with foci in unsynchronised cultures. By contrast, the number of foci per cell (data not shown) is correlated to the fluence and, therefore, to the number of induced breaks, as recently shown for -H2AX (Rothkamm and Lobrich, 2003
).
Protein interactions confirm active DSB repair complexes
To verify whether DSB is activated, and Rad51, DNAPKcs and -H2AX foci represent HRR and NHEJ, co-localisation of the foci together with a selection of other repair proteins of the two pathways were studied after a single exposure to 960 kJ/m2 (30 minutes) without post incubation. Spatial neighbourhood was detected by the co-localisation of pairs of two proteins by laser scanning microscopy. Additionally, close spatial co-localisation was detected by the FRET technique, which detects whether two chromophores (proteins) are in a range of approximately 10 nm or closer (Periasamy, 2001
; Sekar and Periasamy, 2003
; Hink et al., 2002
). We present the co-localisation results of Rad51 (green) and Rad52 (red) (Fig. 4A). Not only an optical co-localisation (arrow) but also FRET signals were detected. This indicates a close neighbouring of the two factors, or even molecular interaction, during HRR. Also the Mre11/Rad50 can be found after UV-A exposure (Fig. 4B) with a positive FRET effect. In addition to the optical detection of spatial co-localisation the interaction of Rad51/Rad52 and Mre11/Rad50 can be found by Co-IP (Fig. 4C). Taken together these results show that DSB repair is activated in response to UV-A irradiation within the 30 minutes of UV-A exposure and that Rad51 foci represent HRR repair sites and the intra-pathway interactions are confirmed.
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Interpathway co-localisation of Rad50/Rad52, Rad51/XRCC4, KU70/Rad51 and DNA-PKcs/Rad51 showed an incomplete optical co-localisation with approximately 20% of the foci co-localised but no FRET signal. Taken together these data suggest that in some, but not all, DSB foci both repair systems are active, but do not have a direct interaction. Table 1 summarises the results from the crosstalk studies. Each protein pair has been studied independently. A minimum of 25 cells per protein pair have been analysed in replicated experiments. The interactions were classified as strong (+), weak (0) or not detectable (-).
Temporal sequence: focus and complex formation
The temporal choreography of the two repair pathways was studied using the focus formation of Rad51, Mre11, -H2AX, DNA-PKcs and XRCC4. The foci were counted in 2x50 cells 1-24 hours after exposure to 980 kJ/m2 UV-A. We plotted the frequency of foci formation for each of the four examined repair proteins (Fig. 5A). The persistence of the foci allows an interpretation of the duration of the repair process. The
-H2AX foci provide fastest signal observed, even after 5 minutes of UV-A exposure a significant increase becomes visible (compare with Fig. 2). After 30 minutes of exposure, foci of Rad51, Mre11, DNA-PKcs and XRCC4 are visible as well. Nevertheless, the number of Rad51 foci continues to increase even after the exposure has ended. The Mre11 and the
-H2AX foci disappear, or are reduced significantly, within the first 5 hours. Also the number of cells with DNA-PKcs foci is reduced, but at a slower rate. The number of cells with
-H2AX foci reaches the control level after 5 hours. The Rad51, DNAPKcs and XRCC4 foci are persistent longer. The control level is reached after 24 hours, although after 5 hours a significant decrease is already observed.
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The formation of protein-protein complexes after UV-A exposure was monitored the same way (Fig. 5B). Because the rate of protein-protein complex formation depends on the slowest partner, the Rad51-containing complexes show a slow formation rate. By contrast, the Mre11/Rad50 complex (as an initiator complex for DSB repair) is formed very quickly, but afterwards is removed nearly completely within 5 hours post irradiation. The Rad51/-H2AX complex is built slowly due to the reduced rate of formation of Rad51 foci and is also quickly disassembled probably due to the dephosphorylation of the
-H2AX (compare with Fig. 5A) The Rad51/Rad52 complex is detectable for 5 hours at elevated levels, which hints to a prolonged phase of HRR repair after an initial fast NHEJ repair event. A sample image series shows the formation of Rad51 foci after UV-A irradiation and the subsequent removal (Fig. 5C). Additionally, we also show the
-H2AX/Rad51 complex during and after UV-A exposure (Fig. 5D). Three hours after exposure the majority of the cells show only Rad51 foci (up to 5 hours, see Fig. 5C), whereas 10 minutes after exposure the number of
-H2AX foci is greater than the number of Rad51 foci.
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Discussion |
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The fastest response found after exposure to UV-A was the phosphorylation of H2AX. Even after small doses, foci become visible within 5 minutes. It has been demonstrated that the -H2AX foci label all DSBs (Rothkamm and Lobrich, 2003
). Because the H2AX phosphorylation is just a modification of existing histones, it is reasonable to assume that there is no saturation limit in the number of foci per cell. By contrast, the subsequently activated repair systems are limited in number by the available proteins. From our results we can conclude that the number of HRR repair sites is exceeded by the number of NHEJ sites, even in G2 cells. Therefore, it can also be concluded that the NHEJ system is active throughout the entire cell cycle in human cells and is not decreased in its activity by the HRR in G2. While the co-localisation data show a recruitment of all examined proteins to DSB sites (i.e. a co-localisation with
-H2AX foci) the interaction between the single repair factors is different (Fig. 6).
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From the temporal sequence of the co-localisation of repair proteins, we can confirm prior data at the protein level (Richardson and Jasin, 2000) (see Fig. S1 in supplementary material). The observation that single factors are recruited to DSB sites leads to the following idea: a DSB is initially marked by the cell with
-H2AX, then the Mre11/Rad50/NBS complex initiates DSB repair with an activation of NHEJ in G1 cells, followed by the recruitment of DNA-PKcs, and then followed by XRCC4, which remains at the DSB site for several hours. In late S or G2, when both systems are available, the initial sequence of
-H2AX focus formation to Mre11/Rad50 complex recruitment is followed by a concerted action of both DSB repair pathways. In our experiments, the Rad51/Rad52 complex is formed after DNA-PKcs recruitment, but concomitant with the XRCC4 foci. Therefore the temporal sequence for DSB repair in G2 can be established as follows: (1) H2AX phosphorylation; (2) Mre11/Rad50 complex formation (probably including NBS1 as well); (3) DNA-PKcs recruitment; and (4) association of Rad51 followed by the Rad51/Rad52 complex formation concomitant with ligation by XRCC4.
From the temporal sequence it can also be concluded that the -H2AX signal is maintained by the cell until all repair factors are loaded onto the DSB site, whereupon the
-H2AX label is removed from the DSB site, while the other repair factors stay at the site. In contrast to recent findings, where persisting
-H2AX foci where found (Rothkamm and Lobrich, 2003
; MacPhail et al., 2003
), we did not observe such foci, but observed a complete repair with an early dephosphorylation of
-H2AX. These contradictory results are possibly due to the different radiation sources used in these studies and, therefore, different DSB structures are formed, which are known to be cell-type specific (MacPhail et al., 2003
). Similarly, the Rad50/Mre11 complex is maintained only during the initiation of the repair, which seems reasonable because the complex is supposed to participate in end processing and stabilisation.
The absolute numbers of DSB (-H2AX), Rad51 and Mre11 foci give the impression that the HRR is nearly always co-localised with the NHEJ. It is unclear whether the number of proteins is limited to form these foci or whether the DNA structure of the DSB excludes the HRR from some DSB sites.
Recent studies on the effects of BRCA mutations have given further hints to a combined activity of HRR and NHEJ. BRCA1 mutations, which affect HRR activation, do not lead to a reduction in repair capacity, but to a reduced quality of the repair. For example, NHEJ can almost completely repair DSBs alone, but HRR may participate as an additional proofreading step, if available. Similar findings have also been reported for the combination of mismatch repair and NHEJ.
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Acknowledgments |
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Footnotes |
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References |
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