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Genes to Cells (2006) 11, 513-524. doi:10.1111/j.1365-2443.2006.00958.x
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Sub-nuclear localization of Rad51 in response to DNA damage

Emil Mladenov1, Boyka Anachkova1 and Irina Tsaneva2,*

1 Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria
2 Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, UK


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The repair of DNA double-strand breaks involves the accumulation of key homologous recombination proteins in nuclear foci at the sites of repair. The organization of these foci in relation to non-chromatin nuclear structures is poorly understood. To address this question, we examined the distribution of several recombination proteins in subcellular fractions following treatment of HeLa cells with ionizing radiation and the crosslinking agent mitomycin C. The results showed association of Rad51, Rad54, BRCA1 and BRCA2, but not Rad51C, with the nuclear matrix fraction in response to double-strand breaks induction. The association of Rad51 with the nuclear matrix correlates with the formation of Rad51 nuclear foci as a result of DNA damage. Fractionation in situ confirmed that Rad51 foci remained firmly immobilized within the chromatin-depleted nuclei. Irs1SF cells that are unable to form Rad51 damage-induced nuclear foci did not show accumulation of Rad51 in the nuclear matrix. Similarly, no accumulation of Rad51 in the nuclear matrix could be observed after treatment of HeLa cells with the kinase inhibitor caffeine, which reduces formation of Rad51 foci. The results were compared to the distribution of the phosphorylated histone variant, {gamma}-H2AX. These data suggest a dynamic association and tethering of recombination proteins and surrounding chromatin regions to the nuclear matrix.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Homologous recombination (HR) is an important repair pathway for the maintenance of genome integrity. It is essential for error-free repair of DNA lesions that affect both DNA strands, such as double-strand breaks and interstrand crosslinks, and is involved in the repair of stalled and broken replication forks occurring naturally during DNA replication. The major HR proteins such as Rad51, Rad52, Rad54 etc., are re-localized within the nucleus in response to DNA damage to form distinct foci that can be visualized by immunofluorescent microscopy and are thought to represent assemblies of proteins at the sites of repair (Haaf et al. 1995; Tan et al. 1999; Liu & Maizels 2000; Essers et al. 2002; Tarsounas et al. 2004).

Rad51 was found to associate with chromatin in response to DNA damage or during DNA replication (Tarsounas et al. 2003), clearly reflecting its recruitment to ongoing recombination reactions. The formation of nuclear foci, however, indicates a higher level of chromatin organization. The mechanisms involved in this process and the relation of foci formation to the underlying nuclear substructures are poorly understood (van Gent et al. 2001; Rouse & Jackson 2002; West 2003). During interphase, chromosomes occupy discrete areas in the nucleus termed chromosome territories. The mechanisms that maintain the chromosome territories involve a non-chromatin compartment referred to as the nuclear matrix, scaffold and nuclear bodies that contain macromolecular complexes for DNA processing (Ma et al. 1999; Cremer et al. 2000). These macromolecular complexes are dynamic; their components are exchanging and their structure depends on and is inseparable from their function (Cook 1999; Stein et al. 2003; Kosak & Groudine 2004). There is compelling evidence that DNA transactions such as transcription and DNA replication are carried out at nuclear foci that are associated with the nuclear matrix. These foci are centers in which coregulated clusters of replicons and genes are simultaneously processed by the replication and transcriptional machinery (Hozak et al. 1993; Jackson & Pombo 1998; Ma et al. 1998; Cook 1999; Radichev et al. 2005). There are data indicating that the nuclear matrix is the site of assembly of nucleotide excision repair complexes. Proteins involved in nucleotide excision repair are relocalized in nuclear foci and the proteins along with the damaged DNA are recruited to the nuclear matrix soon after UV irradiation (Jackson et al. 1994; Balajee et al. 1998; Volker et al. 2001). Similar recruitment may well apply to proteins involved in the HR repair pathway. The BLM helicase, which is thought to function in recombination and repair of stalled replication forks was found to associate with Rad51 and resided in nuclear matrix-associated complexes (Bischof et al. 2001). Biochemical fractionation revealed that Fanconi anemia (FA) proteins, which constitute a pathway that overlaps with homologous recombination (D’Andrea & Grompe 2003; West 2003) localized in chromatin and the nuclear matrix fractions. Treatment with the crosslinking agent mitomycin C (MMC) resulted in the increase of the FA proteins attached to the nuclear matrix (Qiao et al. 2001). Sub-cellular fractionation experiments have shown that the tumor suppressor proteins BRCA1 and BRCA2 that mediate the assembly of HR proteins were also found to interact with the nuclear matrix (Huber & Chodosh 2005). Recently we have shown that after transfection of cells with plasmids damaged in vitro by introduction of interstrand crosslinks, both the damaged DNA and the HR protein Rad51 were specifically associated with the nuclear matrix fraction (Atanassov et al. 2005). These findings suggest a role for the nuclear matrix in the process of DNA recombination.

To answer whether Rad51 focus formation was associated with the nuclear matrix we looked for correlation between the appearance of nuclear foci and the distribution of Rad51 in different subcellular fractions, in response to treatment with ionizing radiation (IR) and MMC. The results showed that while in control cells over 90% of Rad51 was found in the Triton X-100 soluble fraction, treatment with MMC or X-rays brought about a pronounced immobilization of the protein in the Triton X-100 insoluble fraction. Digestion of chromatin with DNase I and extraction with high salt did not remove significant amount of the insoluble Rad51, which remained in the nuclear matrix fraction. Fractionation in situ directly showed that Rad51 foci were attached to the nuclear matrix. The distribution of Rad51 among the different subnuclear fractions was compared to that of Rad54, Rad51C, BRCA1, BRCA2, and {gamma}-H2AX. Under the conditions of our experiments, Rad51C was found free in the nucleosol while the rest of the proteins followed the distribution pattern of Rad51. The results suggest that the assembly of recombination proteins into repair complexes takes place at the nuclear matrix.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
The mammalian Rad51 is the key recombination protein promoting the pairing and exchange of strands between homologous DNA molecules. It functions as a helical nucleoprotein filament (West 2003), which constitutes the core of the HR repair reaction. To test whether the assembly of recombination proteins into nuclear foci is physically associated with the nuclear matrix we searched for correlation between Rad51 foci formation and the association of Rad51 with the nuclear matrix after treatment with agents that induce interstrand crosslinks and double-strand breaks. Exponentially growing HeLa cells were treated with 1 µM MMC for 16 h or irradiated with 7.5 Gy of X-ray and incubated for 2 h at 37 °C. Examination of the cells by immunofluorescent microscopy showed that both treatments resulted in the formation of prominent Rad51 foci. The percentage of Rad51 foci positive cells in the untreated control was 15%, while about 50% of the MMC-treated cells and 60% of the irradiated cells developed Rad51 nuclear foci. Samples of the control and treated cells were subjected to the following biochemical fractionation and isolation of nuclear matrix. The cells were lyzed by treatment with Triton X-100. The Triton insoluble nuclear fraction was digested with DNase I, and the digested chromatin extracted with buffer containing 0.1 M (NH4)2SO4 (DNase I soluble fraction). The DNase I resistant nuclear fraction was further extracted with 0.65 M (NH4)2SO4-containing buffer to obtain the high salt soluble fraction and the high salt insoluble fraction representing the nuclear matrix. Equal amounts of proteins from each fraction were fractionated by SDS polyacrylamide gel electrophoresis, transferred on to nitrocellulose membranes and immunostained with anti-Rad51 antibodies. Anti-ß-actin staining was used as a loading control. Staining with anti-{alpha}-tubulin and anti-histone H3 antibodies was used to monitor the fractionation procedure (Fig. 1A). Triton X-100 treatment efficiently removed cytoplasmic components, as seen by staining for {alpha}-tubulin. Treatment with 0.65 M (NH4)2SO4 resulted in the efficient extraction of proteins bound to DNA by strong non-specific electrostatic interactions such as the histones. The Western blots of the fractions stained with anti-Rad51 antibodies (Fig. 1B) were quantified by scanning and densitometry (Fig. 1C). The results show that DNA damage resulted in a significant decrease of Rad51 in the Triton X-100 soluble fraction with a concomitant increase of Rad51 in the Triton X-100 insoluble nuclear fraction. While in the untreated cells over 90% of Rad51 was found in the Triton X-100 soluble fraction, this percentage decreased to 50% after treatment with MMC and 35% after X-rays irradiation (P-values less than 0.005). DNase I digestion and extraction with 0.1 M ammonium sulfate released only about 10% of the chromatin-associated Rad51 while the majority of Rad51 remained DNase I resistant. Further extraction with 0.65 M ammonium sulfate did not release Rad51 in soluble form and over 35% of Rad51 remained in the nuclear matrix fraction after MMC treatment (P-value 0.001). Similarly, more than 50% of Rad51 remained in the nuclear matrix fraction after X-ray irradiation (P-values < 0.001). The amount of matrix-associated Rad51 in the cells treated with MMC was 7 times higher, compared to non-treated cells. This increase was even more pronounced in the irradiated cells where the amount of matrix-attached Rad51 was 10 times higher than in control cells.


Figure 1
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  		Figure 1  Sub-cellular fractionation of Rad51 in HeLa cells after treatment with MMC and ionizing radiation. The cells were treated with 1 µM MMC for 16 h or 7.5 Gy X-rays, and subjected to biochemical fractionation as described in Experimental procedures into Triton X-100 soluble and insoluble fractions, DNase I soluble and DNase I resistant fractions, and high salt soluble and nuclear matrix fractions. Equal amounts of protein from each fraction were resolved by SDS polyacrylamide gel electrophoresis, transferred on to nitrocellulose membranes and incubated with the antibodies. The secondary antibodies were anti-rabbit IgG antibody conjugated with AlexaFluor800 and anti-mouse IgG antibody conjugated with AlexaFluor700. (A) Immunostaining with anti-{alpha}-tubulin and anti-histone H3. (B) Immunostaining with anti-Rad51-Ab1 antibody. (C) Densitometric analysis of the distribution of Rad51 among the subcellular fractions. The nitrocellulose membranes presented in Figure 1B were visualized and scanned by Odyssey infrared imaging system. After normalization of the amount of Rad51 against the loading control (ß-actin), the distribution of Rad51 in the subcellular fractions was determined. The amount of Rad51 was expressed as percentage of the total amount of Rad51 in the Triton X-100 soluble and insoluble fractions taken as 100%. The results are means of three independent experiments and the standard deviations are shown with vertical bars.

 
These results correlated with the increased percentage of Rad51 nuclear foci-positive cells obtained after treatment and suggested that the observed accumulation of Rad51 in the nuclear matrix fraction was due to Rad51 assembled in foci formed in response to DNA damage. In order to test this suggestion directly we examined the process at the cellular level. Control, MMC-treated and irradiated HeLa cells were extracted in situ as described in Experimental procedures, and immunostained with anti-Rad51 antibodies (Fig. 2). Extraction with 0.5% Triton X-100 had little effect on the Rad51 foci (Fig. 2B). Control experiments showed efficient removal of {alpha}-tubulin by this treatment but retention of laminB staining under the same conditions, as expected (Supplementary Fig. S1). Treatment with DNase I and extraction with high salt resulted in the complete loss of DNA fluorescence as shown by staining with DAPI (Fig. 2C–F). However, while no Rad51 staining was detectable in the control cells after the in situ fractionation (Fig. 2F), more than 50% of the damage-induced Rad51 foci could be detected in the nuclear matrix in irradiated or MMC-treated cells (Fig. 2D,E). This percentage may even be higher, since during the extraction procedure, Rad51 immunoreactivity decreased significantly, thus making the detection of foci difficult due to their diminished intensity. Recently Huber & Chodosh (2005) reported that irradiation-induced Rad51 nuclear foci were not observed in the nuclear matrix following in situ fractionation. The differences between their results obtained by the amine modification method for in situ isolation of nuclear matrix and the results presented above may be due to the method of fractionation, the use of a different cell line and/or to the antibodies used.


Figure 2
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  		Figure 2  Rad51 foci are associated with the nuclear matrix. HeLa cells were irradiated with 7.5 Gy X-rays (A) and subjected to in situ fractionation as described in Experimental procedures into (B) Triton X-100 insoluble, (C) DNase I resistant and (D) nuclear matrix fractions. (E) The nuclear matrix fraction from HeLa cells treated with 1 µM MMC. (F) The nuclear matrix fraction obtained after in situ fractionation of control, untreated HeLa cells. The cells were stained with mouse anti-Rad51 antibody, followed by staining with anti-mouse IgG antibody conjugated with FITC. DNA was stained with DAPI.

 
To confirm the suggestion that the assembly of Rad51 foci is associated with the nuclear matrix, we performed biochemical fractionation on a cell line, in which damage-induced Rad51 foci do not form (irs1SF). The assembly of Rad51 foci in response to DNA damage depends on a group of proteins known as Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3) (Thacker 1999). The Rad51 paralogs are structurally related to each other and share 20–30% similarities with Rad51. They are thought to act as accessory factors for the assembly of the Rad51 synaptic filament. The irs1SF cells are a Chinese hamster ovary cell line, deficient in XRCC3. Irs1SF cells are sensitive to DNA damaging agents, defective in homology-directed DNA repair and do not form damage-induced Rad51 foci (Bishop et al. 1998; Liu et al. 1998; Pierce et al. 1999). Recent data have shown that XRCC3 associates directly and independently of Rad51 with DNA damaged sites, as judged by its co-localization with {gamma}-H2AX foci, and subsequently facilitates the formation of Rad51 repair complexes (Forget et al. 2004). The results of the biochemical fractionation of irs1SF cells are presented on Fig. 3A. The distribution of Rad51 among different fractions from the parental wild-type CHO-AA8 cells was similar to that of HeLa cells. After treatment with MMC Rad51 was found predominantly in the nuclear matrix fraction and the increase in the amount of matrix-associated Rad51 was approximately seven-fold in comparison with the untreated cells. In the XRCC3-deficient derivative cell line irs1SF no increase in the matrix-associated Rad51 could be detected and Rad51 remained predominantly in the Triton X-100 soluble fraction.


Figure 3
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  		Figure 3  Sub-cellular fractionation of Rad51 in cells with reduced damage-induced Rad51 focus formation. (A) Wild-type Chinese hamster cells (CHO-AA8) and its derivative irs1SF cells deficient in XRCC3 were treated with 1 µM MMC and then subjected to biochemical fractionation and immunoblotting as in Fig. 1. (B) HeLa cells treated with 1 µM MMC and 8 mM caffeine (CF) for 16 h and subjected to biochemical fractionation and immunoblotting as in Fig. 1. The secondary antibodies were anti-mouse IgG antibody conjugated with alkaline phosphatase and anti-mouse IgG antibody conjugated with horse-radish peroxidase.

 
Genomic stability in cells that have suffered DNA damage is maintained by the activation cell cycle checkpoints that delay cell cycle progression and induce DNA repair. To elucidate whether the association of Rad51 with the nuclear matrix is under the control of the cell cycle checkpoints, HeLa cells were treated with MMC in the presence of the protein kinase inhibitor caffeine. The latter overcomes cell cycle checkpoint responses, inhibits repair of double-strand breaks by HR and significantly reduces formation of damage-induced Rad51 foci as shown by immunofluorescence microscopy (Wang et al. 2004; Sorensen et al. 2005). After treatment with caffeine, the cells were subjected to subcellular fractionation and the distribution of Rad51 in the fractions was determined by Western blotting. The results show (Fig. 3B) that in the caffeine-treated cells Rad51 remained predominantly free in the nucleosol and there was a pronounced reduction in the fraction of Rad51 immobilized to the nuclear matrix fraction. This result suggests that the association of Rad51 with the nuclear matrix is under cell cycle checkpoint control and is a part of the normal physiological response to DNA damage.

Taken together, these results show that the accumulation of Rad51 in the nuclear matrix fraction is due to the formation of nuclear foci and suggests that damage induced Rad51 foci are stably associated with a non-chromatin nuclear component. It may be that the assembly of Rad51 repair factories involves interactions with the nuclear matrix and thus the HR reaction is tethered to the nuclear matrix. Alternatively, it may be that Rad51 cofractionates with the nuclear matrix fraction because it forms synaptic filaments, which would protect DNA from DNase I digestion. To examine more carefully the association of Rad51 with the nuclear matrix, nuclei were incubated with increasing concentrations of DNase I, ranging from 10 to 160 U/mL, and subsequently extracted with low and high salt buffers (Fig. 4). The results show that the amount of Rad51 in the nuclear matrix fractions was not affected by the degree of chromatin digestion by DNase I. Even the highest concentration of DNase I used in these experiments (producing about 5% of matrix-attached DNA in the form of fragments less than 200 bp long) did not release Rad51. These results indicate that the association of Rad51 with the nuclear matrix fraction did not depend on the integrity of DNA but most likely involved protein-protein interactions.


Figure 4
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  		Figure 4  The amount of Rad51 associated with the nuclear matrix fractions is not affected by the degree of chromatin digestion by DNase I. Cells were treated with 1 µM MMC. After extraction with Triton X-100, the nuclei were incubated with increasing concentrations of DNase I as indicated, and subsequently extracted with low and high salt buffers. The nuclear matrix fractions were subjected to Western blotting as in Fig. 1. The secondary antibody was anti-mouse IgG conjugated with alkaline phosphatase.

 
To test whether HR repair complexes are assembled at the nuclear matrix, we followed the localization of other proteins involved in the recombination reaction. To this end, we probed the subcellular distribution of Rad51C, Rad54, BRCA1 and BRCA2. Rad51C was chosen as an example of a HR protein for which there were no data for co-localization with Rad51 in nuclear foci, while Rad54, BRCA1 and BRCA2 are proteins that co-localize with Rad51 in damage-induced foci (Chen et al. 1999; Scully et al. 1997). Different subcellular fractions obtained from HeLa cells before or after treatment with IR were immunostained with anti-Rad51C (Fig. 5A). The results show that Rad51C was found in the Triton X-100 soluble fraction only and no changes in the distribution of Rad51C could be observed after IR treatment. Cell lysis in hypotonic buffer in the absence of Triton X-100 showed that Rad51C was found in the nucleus (data not shown). While the absence of Rad51C from damage-induced Rad51 nuclear foci could be due to Rad51C concentrations being too low to detect by immunofluorescent microscopy, our biochemical data show that Rad51C did not form stable associations with structures in the nucleus. The results are in agreement with immunoprecipitation data showing that Rad51 was excluded from Rad51C-containing complexes with Rad51B, Rad51D, XRCC2, and XRCC3 (Miller et al. 2002). Rad54, BRCA1 and BRCA2 were found to accumulate in the nuclear matrix fraction in response to DNA damage and this accumulation closely paralleled the recruitment of Rad51 to this fraction (Fig. 5). Rad54 is supposed to take part in the invasion step of the homologous recombination reaction (West 2003). Its damage-induced recruitment to the nuclear matrix fraction was less pronounced than that of Rad51 (Fig. 5A). This is consistent with the dynamics of Rad54 in nuclear foci studied by fluorescence recovery after photobleaching (FRAP) showing that Rad54 is more mobile and less tightly attached within the foci than Rad51 (Essers et al. 2002). The results for BRCA1 and BRCA2 are in full agreement with recent data showing the association of these proteins with the nuclear matrix (Huber & Chodosh 2005). Nevertheless, it could be argued that BRCA1 and BRCA2 are very large proteins, which could be refractory to the extraction procedures. However, the results clearly showed Triton-soluble and high-salt soluble BRCA1 population (Fig. 5B). Therefore, BRCA1 could be extracted under the experimental conditions used and its association with the nuclear matrix fraction indicates immobilization through some stable interactions with the repair foci. It should be noted that the interaction between BRCA1 and Rad51 is mediated through BRCA2, which interacts directly with Rad51 through a series of degenerative motifs known as BRC repeats (West 2003) and is supposed to play a role in the delivery of Rad51 to the damaged sites (Pellegrini & Venkitaraman 2004).


Figure 5
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  		Figure 5  Comparison of the subcellular distribution of Rad51 with that of Rad51C, Rad54, BRCA1, and BRCA2. The cells were treated with ionizing radiation and then subjected to biochemical fractionation as in Fig. 1. (A) Western blot analysis with antibodies against Rad51, Rad51C and Rad54. (B) The proteins were resolved by gradient SDS polyacrylamide gel electrophoresis and Western blot analysis performed with antibodies against BRCA1 and BRCA2. Secondary antibodies were conjugated with horse-radish peroxidase.

 
There are data that chromosomes perform movements within the chromosome territories in response to DNA damage (Aten et al. 2004). It could be envisaged that regions containing damaged DNA may become attached to subnuclear structures as a result of chromatin remodeling. To test this possibility we examined the subnuclear distribution of the phosphorylated histone variant {gamma}-H2AX, which marks chromatin regions that have suffered double-strand breaks and will be engaged in the repair process (Rogakou et al. 1998). For biochemical analysis, HeLa cells were collected 2 h after X-ray irradiation and the cells were fractionated as described. In untreated control cells, the Western blot analysis using anti-{gamma}-H2AX antibodies showed very low, barely detectable signal in the Triton X-100 insoluble fraction. Treatment of the cells with ionizing radiation resulted in the appearance of a prominent band of {gamma}-H2AX in this fraction. Further fractionation by DNase I digestion and extraction with low and high salt buffer showed release of {gamma}-H2AX in the chromatin fractions (DNase I soluble and high salt-soluble) but interestingly, a significant {gamma}-H2AX signal remained in the nuclear matrix even after high salt extraction (Fig. 6). This association with the nuclear matrix seems specific for the phosphorylated histone variant, because only negligible amounts of unmodified histones were found in the nuclear matrix fraction (Fig. 1A). The results suggest that chromatin regions in close proximity to the double-strand breaks may become attached to the nuclear matrix.


Figure 6
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  		Figure 6  Sub-cellular distribution of histone {gamma}-H2AX in HeLa cells. Cells irradiated with 7.5 Gy X-ray were subjected to biochemical fractionation and Western blotting as in Fig. 1. The blots were stained with rabbit anti-{gamma}-H2AX and mouse anti-Rad51 antibodies. The secondary antibodies were anti-rabbit IgG antibody conjugated with AlexaFluor800 and anti-mouse IgG antibody conjugated with AlexaFluor700.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Redistribution of Rad51 in response to DNA damage was shown previously using biochemical fractionation and was attributed to the recruitment of Rad51 to chromatin (Tarsounas et al. 2003). Taking the fractionation procedure further, we show that Rad51 accumulated in the fraction that was resistant to DNase I digestion and extraction with 0.65 M ammonium sulfate (nuclear matrix). Similarly, we also show accumulation of Rad54, BRCA1 and BRCA2 in this fraction after DNA damage. The association of these proteins with the nuclear matrix fraction correlated tightly with the formation of Rad51 nuclear foci. Accumulation of Rad51 in the nuclear matrix did not occur in irs1SF cells that are unable to form damage-induced nuclear foci, or in HeLa cells treated with caffeine, which abrogates formation of damage-induced Rad51 foci. Fractionation of the cells in situ showed directly that a significant proportion of the Rad51 foci remained in the chromatin-depleted cells after DNase I digestion and extraction with high salt. All these results support the conclusion that the biochemical fraction obtained following a routine procedure for isolation of nuclear matrix contains the repair foci observed microscopically. The fractionation procedure therefore yields the Rad51 population that is actually recruited to the repair process. The structural basis for the fractionation behavior of Rad51, Rad54, BRCA1 and BRCA2 in response to DNA damage is not clear and cannot be defined by our experiments. However, understanding the physical associations involved could help to understand the organization and control of Rad51 repair foci that assemble in response to DNA damage. Furthermore, the fractionation procedure used in these experiments could prove to be a useful approach towards identifying the role of post-translational modifications for the assembly and activation of the repair process.

Regardless of some controversy that surrounds research on the nuclear matrix (Hancock 2004; Zaidi et al. 2005) there is a wealth of evidence suggesting that all DNA transactions involve some sort of organization and association with underlying nuclear structures. One specific structure that needs to be considered first is the assembly of Rad51 into synaptic nucleoprotein filaments, which is consistent with its immobile state in vivo (Essers et al. 2002). However, the formation of synaptic filaments alone cannot fully explain the fractionation of Rad51 with the nuclear matrix. The concentration of Rad51 protein in damage-induced foci must be very high for the structures to be observed microscopically. It is difficult to estimate how much of Rad51 is engaged in nucleoprotein filaments undergoing repair but it seems unlikely that it would involve all Rad51 accumulated in the foci. Moreover, neither BRCA1 nor BRCA2 would be a physical part of the Rad51 filaments. In addition, while filament formation would protect the DNA inside the filament against DNase I digestion, these filaments as a whole (or at least some of them) should be released from chromatin by nuclease digestion. However, we observed little or no release of soluble Rad51 from the nuclei even after extensive nuclease digestion. As DNase I digestion did not affect the amount of Rad51 associated with the nuclear matrix this indicates that in the context of nuclear microenvironment or subnuclear compartments (Zaidi et al. 2005), this tethering would be protein-based rather than DNA-based.

It has been demonstrated that damaged plasmid DNA transfected in cells is recruited to the nuclear matrix to be repaired by the HR pathway (Atanassov et al. 2005). The association with the nuclear matrix of a fraction of {gamma}-H2AX, which marks chromatin regions that have suffered double-strand breaks, confirm that finding. The accumulation of HR proteins to the nuclear matrix fraction in response to DNA damage suggests that repair centers are assembled at the nuclear matrix and damaged DNA is recruited to these centers rather than the recombination proteins being assembled directly on DNA at the site of damage. The association of the damaged DNA to the nuclear matrix-associated recombination protein complexes can be attained by the small-scale movements of subchromosomal domains containing double-strand breaks (Aten et al. 2004). The nuclear matrix could be regarded as a dynamic entity that reflects a specific physiological response rather than a rigid skeleton (Zaidi et al. 2005). The assembly of the repair complexes and their immobilization in the nucleus could represent such a dynamic association. Tethering of the repair foci could be regarded as an integral part of the dynamic chromatin reorganization in response to DNA damage.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cells and treatment

HeLa M cells were grown as monolayer in Dulbecco's modified Eagle's medium. Chinese Hamster ovary (CHO) wild-type cells AA8 and its derivative cell line irs1SF (XRCC3 deficient) were cultured in alpha-MEM medium. The media were supplemented with 10% fetal bovine serum and antibiotics and the cells were grown in an atmosphere of 95% air, 5% CO2 at 37 °C. Exponentially growing cells were treated with 1 µM mitomycin C from Streptomyces caespitosus for 16 h. Caffeine was used at a concentration of 8 mM. For X-ray irradiation, exponentially growing cells were irradiated with 7.5 Gy with high-voltage X-ray irradiator at a rate of 2.5 Gy per minute for 3 min and incubated at 37 °C for 2 h after the treatment.

Cell fractionation

Cells were collected with cell scraper, washed in PBS (0.14 M NaCl, 0.01 M phosphate buffer, pH 7.0). 1–2 x 107 cells were suspended in 1 mL CSK buffer (0.5% Triton X-100, 100 mM NaCl, 3 mM MgCl2, 300 mM sucrose, 1 mM EGTA, 10 mM PIPES pH 6.8) containing protease inhibitors (complete inhibitors, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail 1 and 2, Sigma) and incubated for 10 min on ice. The cells were spun down at 500 g for 5 min at 4 °C and the supernatant and pellet were collected. They were designated as the Triton X-100 soluble and Triton X-100 insoluble fractions, respectively. The Triton soluble fraction was clarified by additional centrifugation at 15 000 g for 10 min at 4 °C. The Triton insoluble fraction was washed with buffer A (10 mM NaCl, 5 mM MgCl2, 250 mM sucrose, 1 mM EGTA, 10 mM Tris-HCl, pH 7.6) containing protease and phosphatase inhibitors and resuspended in 200 µL of the same buffer. Eighty µ/mL of RNase free DNase I (Roche) was added and the nuclei were incubated for 30 min at 37 °C. After the incubation the suspension was spun down at 700 g for 5 min at 4 °C. The pellet was extracted with 100 µL of 0.1 M ammonium sulfate by incubation for 10 min on ice and centrifugation at 700 g for 5 min at 4 °C. The supernatant was indicated as DNase I soluble fraction. Aliquot of the pellet was suspended in buffer A, containing 8 M urea and indicated as the DNase I resistant fraction. Another aliquot of the pellet was extracted twice with 50 µL of 0.65 M ammonium sulfate by incubation for 10 min on ice and centrifugation at 700 g for 5 min at 4 °C. The supernatant was indicated as high salt soluble fraction. The pellet was dissolved in buffer A, containing 8 M urea and indicated as the high salt insoluble fraction.

For in situ fractionation and immunofluorescent microscopy, the cells were grown on cover slips in 24 well plates. They were fractionated in situ by washes following the scheme described above. Briefly, the cells were washed with CSK buffer followed by buffer A and incubated for 30 min with 80 µ/mL DNase I in buffer A. After washes with low and high salt buffer (0.1 and 0.65 M ammonium sulfate in buffer A, respectively) the cells were fixed with 2% paraformaldehyde (PFA) and processed for immunofluorescent microscopy as described below.

Electrophoresis and Western blot analysis

Electrophoresis was carried in 12.5% gels and in 4% to 12.5% polyacrylamide gradient gels according to Laemmli (1970). For Western blot analysis the proteins were transferred on to 0.45 µM nitrocellulose membranes (Trans-blot transfer medium, Bio-Rad) using a semidry Western blot device following the instructions by the manufacturer (Transblot SD, Bio-Rad). Equal loading and transfer was monitored by Ponceau S staining of the membrane and actin immunoblotting. After the transfer the membranes were incubated in blocking buffer (5% semi dry milk in TBS-T (0.1% Tween 20, 150 mM NaCl, 25 mM Tris-HCl, pH 7.6)) for minimum 1 h at room temperature or overnight at 4 °C, after which the membranes were incubated for 1 h with the primary antibody, washed 3 times for 10 min with TBS-T and incubated for 1 h with the secondary antibody. The following antibodies were used: anti-Rad51 (mouse, monoclonal, Abcam); anti-Rad51-Ab1 (rabbit, polyclonal, Oncogene research); anti-{alpha}-tubulin (mouse, monoclonal, Sigma); anti-ß-actin (mouse, monoclonal, Sigma); anti-Rad51C (mouse, monoclonal, Abcam); anti-Rad54 (mouse, monoclonal, Abcam); anti-BRCA1 (mouse, monoclonal, Oncogene research); anti-BRCA2 (rabbit, polyclonal, Oncogene Research); anti-{gamma}-H2AX (rabbit, polyclonal, Abcam); anti-mouse horse-radish peroxidase conjugated (Amersham biosciences); anti-rabbit horse-radish peroxidase conjugated (Amersham biosciences); anti-mouse alkaline phosphatase (Abcam); anti-mouse Alexa-Fluor700-conjugated (Molecular Probes); anti-rabbit AlexaFluor800-conjugated (Molecular Probes). For the last two antibodies, development was carried out with Odyssey infrared imaging system (LI-COR, Biosciences). The rest of the antibody reactions were developed by ECL plus Western Blotting Detection System (Amersham Biosciences) as recommended by the manufacturers. The membranes were visualized by Fuji LAS-1000 CCD camera (Dark Framed –25 °C) with Fuji LAS Software.

Immunofluorescent microscopy

Intact cells and cells fractionated in situ were fixed with 2% paraformaldehyde for 5 min at room temperature. The cells were incubated in 0.3% Tween-20 in PBS for 15 min. After the incubation the slides were blocked in blocking buffer (3% goat serum, 0.3% bovine serum albumin in PBS) for minimum 1 h at room temperature and then incubated in primary antibody diluted in blocking buffer overnight at 4 °C. After washing, the secondary antibody was added and the slides were incubated for at least 1 h. The following antibodies were used: Anti-Rad51 (mouse, monoclonal, Abcam), anti-mouse IgG conjugated with FITC (DAKO Cytomation). The slides were mounted in mounting media (Vectashield mounting media for fluorescence) containing 2 µg/mL DAPI (Vector Laboratories). The slides were observed at AxioPlan2 fluorescent microscope (Carl Zeiss). Grayscale pictures were taken with AxioCam HRc camera (Carl Zeiss) and additionally pseudo colored. The software for capturing the picture was AxioVision v3.1 (Carl Zeiss vision GmbH).


   Acknowledgements
 
We thank Professor J. Thacker, MRC Radiation and Genome Stability Unit, Harwell, for providing the irs1SF cells, Dr S. C. West from Cancer Research UK for gift of antibodies. This work was supported by the Wellcome Trust Collaborative Research Initiative Grant 070397.


   Footnotes
 
Communicated by: Nic Jones

* Correspondence: E-mail: tsaneva{at}biochem.ucl.ac.uk


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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