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Article |
RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation
Correspondence to Madalena Tarsounas: madalena.tarsounas{at}rob.ox.ac.uk
The RAD51 paralogues act in the homologous recombination (HR) pathway of DNA repair. Human RAD51C (hRAD51C) participates in branch migration and Holliday junction resolution and thus is important for processing HR intermediates late in the DNA repair process. Evidence for early involvement of RAD51 during DNA repair also exists, but its function in this context is not understood. In this study, we demonstrate that RAD51C accumulates at DNA damage sites concomitantly with the RAD51 recombinase and is retained after RAD51 disassembly, which is consistent with both an early and a late function for RAD51C. RAD51C recruitment depends on ataxia telangiectasia mutated, NBS1, and replication protein A, indicating it functions after DNA end resection but before RAD51 assembly. Furthermore, we find that RAD51C is required for activation of the checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage. This suggests that hRAD51C contributes to the protection of genome integrity by transducing DNA damage signals in addition to engaging the HR machinery.
© 2009 Badie et al.
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A central role in the process of HR is played by the RAD51 recombinase (West, 2003). In mammalian cells, a recently characterized family of proteins known as the RAD51 paralogues consists of five proteins (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) related to each other and to RAD51 itself. Their precise cellular function remains unknown. Cells lacking the RAD51 paralogues are defective in gene targeting and exhibit a genomic instability phenotype and high sensitivity to ionizing radiation (IR) and DNA cross-linking agents, which are indicative of their role in recombinational DNA repair (Liu et al., 1998; Takata et al., 2001; French et al., 2002). The paralogues form complexes with each other (Masson et al., 2001; Miller et al., 2002; Wiese et al., 2002). Specific subcomplexes are thought to perform distinct cellular functions. RAD51C and XRCC3 but not the other paralogues are part of a complex that possesses branch migration and Holliday junction resolution activities (Liu et al., 2004, 2007). RAD51D, another member of the RAD51 paralogue family, associates with telomeres in meiotic and somatic cells, and its capping function at telomeres is essential for telomere integrity (Tarsounas et al., 2004b). In addition, a role for RAD51C, XRCC2, and XRCC3 in chromosome segregation and centrosome integrity has been proposed (Griffin et al., 2000; Renglin Lindh et al., 2007).
A prominent cytological consequence of DNA damage and checkpoint activation is the accumulation of DNA repair and signaling proteins at the sites of damage that can be visualized microscopically as nuclear foci. In response to IR, RAD51 assembles at DSB sites with well-defined kinetics and molecular requirements (Haaf et al., 1995; Scully et al., 1997). The formation and stability of RAD51 foci is dependent on the presence of BRCA2 and the five RAD51 paralogues (Yuan et al., 1999; Takata et al., 2001; French et al., 2002; Godthelp et al., 2002; Tarsounas et al., 2003, 2004a). This observation, together with the ability of RAD51 to directly interact with both RAD51C and BRCA2 (Chen et al., 1998b; Wong et al., 1997; Rodrigue et al., 2006), led to the concept that these proteins function as cofactors for RAD51 assembly after ssDNA resection at DSB sites. However, although the role of BRCA2 as RAD51 loader is supported by both biochemical and cell biological data, the association of the RAD51C–XRCC3 complex with Holliday junction processing activities suggested that the latter proteins may fulfill a role in the late steps of HR, downstream of RAD51. How RAD51C acts during the early steps of HR and how it promotes RAD51 assembly remain unknown.
In this study, we report that human RAD51C (hRAD51C) is recruited to damage sites in response to treatment with IR or the replication inhibitor hydroxyurea (HU). The kinetics of RAD51C recruitment is consistent with both an early and a late role in HR. We further identify an early function of RAD51C in DNA repair in facilitating phosphorylation of the checkpoint kinase CHK2 and thereby transduction of the damage signal, leading to cell cycle arrest and HR activation. Our data identify RAD51C as a DNA damage response factor required for the activation of S and G2/M checkpoints, which could contribute to the early steps of recombinational DNA repair.
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To confirm this localization pattern, we established single-cell clones stably expressing N-terminally GFP-tagged hRAD51C (Rodrigue et al., 2006) at levels comparable with the endogenous protein. In response to partial irradiation of nuclei with ultrasoft x rays (
2 Gy averaged over the cell), GFP-RAD51C accumulated into foci that overlapped with RAD51 (Fig. S2 B). Preferential localization of the GFP-RAD51C foci in broad stripes within the nuclei, generated by the x-ray mask, further confirms its accumulation at sites of DNA damage.
RAD51C foci colocalize with RAD51 but persist during DSB repair
RAD51C is required for DSB repair by HR, and a direct protein–protein interaction of RAD51C with the RAD51 recombinase has been reported (Rodrigue et al., 2006). This suggested that the two proteins act together in the HR pathway. To investigate whether this was the case, irradiated HeLa cells were costained with mouse anti-RAD51C and rabbit anti-RAD51 antibodies (Fig. 1, E and F). Some of the cells contained mainly RAD51C foci (Fig. 1 E, red circle) or RAD51 foci (Fig. 1 E, green circle), whereas others contained both types of foci that partly colocalized (65% of RAD51C foci colocalized with RAD51, as determined by visual inspection; Fig. 1 E, yellow circle). This pattern suggests that RAD51C and RAD51 play overlapping but not identical roles in response to irradiation.
Detection of cells showing exclusively RAD51C or RAD51 staining and of cells in which both proteins colocalized suggested that the two activities assemble sequentially at the sites of damage. To test this, we monitored the assembly of RAD51C and RAD51 foci in response to radiation-induced DNA damage in a time course experiment (Fig. 2 A). RAD51 is known to form spontaneous foci even in the absence of DNA damage, which is a feature not shared by RAD51C (Fig. 1 B). These foci are thought to represent stalled replication forks during cell cycle progression through S phase (Tarsounas et al., 2003). The blue line in Fig. 2 A indicates the frequency of spontaneous RAD51 foci in nonirradiated control cells. This value was subtracted from the total number of RAD51-positive cells after irradiation. At early time points after irradiation, RAD51C and RAD51 foci appeared with similar kinetics. Although RAD51 foci peaked at 1 h after treatment, RAD51C-positive cells further increased in number and persisted until 6 h after damage. Representative images in Fig. 2 B illustrate these changes. To further characterize the relative kinetics of appearance of the two types of foci, we computationally determined the number of foci in the nuclei of a population of cells (see Materials and methods; Fig. 2 C). This analysis revealed that the number of RAD51C foci per nucleus increased over time and persisted even 6 h after irradiation (Fig. 2 D). In contrast, the number of RAD51 foci per cell already reached a peak 40 min after irradiation and decreased over time (Fig. 2 E), with kinetics similar to that of manually scored RAD51-positive cells (Fig. 2 A). These data are consistent with the possibility that RAD51C acts both at the early stages of DSB processing as well as after RAD51-mediated HR reactions have been completed.
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RAD51C foci form in S or G2 phase
During the aforementioned experiments, we noticed that not all cells exposed to irradiation developed visible RAD51C foci. This could indicate that RAD51C recruitment to DSBs occurs during certain stages of the cell cycle but not others. As HR is known to act predominantly during S and G2, we used centromeric protein F (CENP-F) staining to identify these cells. CENP-F has a nuclear diffuse distribution from S phase onwards, becomes more intense in G2 phase (Fletcher et al., 2003), and shows specific concentration at kinetochores during mitosis. The appearance of nuclear RAD51C foci followed a similar pattern in U2OS cells. Radiation-induced foci were detectable in cells displaying weak CENP-F staining (Fig. 4 A) and reached greater intensity in cells with strong CENP-F. This suggests that RAD51C foci specifically form during S or G2 phase but not G1 phase of the cell cycle.
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The MRN complex is thought to activate ATM at DNA break sites. Therefore, we examined RAD51C recruitment to sites of DNA damage in NBS1-deficient Nijmegen breakage syndrome fibroblasts. Nijmegen breakage syndrome cells complemented with full-length NBS1 were proficient in RAD51C focus formation in response to IR, but cells lacking NBS1 were not (Fig. 4, D and E). These results indicate that the recruitment of RAD51C requires both functional ATM and NBS1. We also visualized RPA foci as a control in these experiments and confirmed that ATM and NBS1 are required for radiation-induced RPA assembly at DSBs (Jazayeri et al., 2006). This indicated that RAD51C is regulated by the ATM pathway, similarly to RPA.
RAD51C accumulation at damage sites depends on RPA
The ATM and NBS1 dependence of both RAD51C and RPA recruitment to damage sites prompted us to test whether both proteins colocalized in response to DNA damage. Indeed, RAD51C and RPA foci closely overlapped in all cells after both irradiation and release from HU treatment (Fig. 5 A). RPA binds ssDNA generated during MRN complex–dependent strand resection. To see whether RAD51C affects strand resection, we analyzed RPA foci formation in cells depleted of RAD51C. RPA assembled normally in response to irradiation, both in cells treated with RAD51C and control siRNAs (Fig. 5 B). As a control, formation of RAD51 foci was efficiently prevented by RAD51C-depletion under our conditions, as previously reported (French et al., 2002; Rodrigue et al., 2006). Conversely, down-regulation of RPA expression completely abolished RAD51C foci formation (Fig. 5, C and D). These data establish that RAD51C colocalizes with RPA and requires RPA binding to ssDNA for its recruitment. Therefore, RAD51C is likely recruited to sites of DSBs after ATM- and NBS1-dependent strand resection and RPA association but before RAD51 filament assembly. How RPA promotes RAD51C recruitment is currently not known. Coimmunoprecipitation experiments did not provide evidence for a direct interaction between the two proteins (Fig. S4).
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RAD51C is a component of three distinct RAD51 paralogue complexes in human cells. A complex formed by RAD51C and XRCC3 has been shown to interact with RAD51 (Rodrigue et al., 2006). Although biochemical analyses suggested that this complex acts in the late stages of HR when Holliday junction intermediates are formed, XRCC3 was also reported to assemble at sites of DNA breaks before RAD51 (Forget et al., 2004). Thus, we tested whether XRCC3 is also required for CHK2 phosphorylation in response to DNA damage. Depletion of XRCC3 using two different siRNAs diminished CHK2 phosphorylation in response to 1 Gy of IR, similarly to RAD51C depletion (Fig. 7 D). This suggests that XRCC3 together with, and probably in complex with, RAD51C promotes checkpoint activation at sites of DSBs. In a similar experiment, depletion of RAD51 did not compromise CHK2 phosphorylation (Fig. S5 C). This is consistent with the possibility that RAD51C and XRCC3 function in CHK2 activation independently of RAD51 filament assembly. CHK2 phosphorylation was detected in nonirradiated RAD51-depleted cells, confirming a DNA repair defect in these cells.
RAD51C foci formation in response to IR was unaffected in Chk2–/– cells (Fig. 7 E), indicating a role for RAD51C upstream of this checkpoint kinase. These results suggest that RAD51C is required for ATM-dependent phosphorylation and activation of CHK2. In turn, reduced CHK2 activation could explain the compromised ability of RAD51C-deficient cells to arrest cell cycle progression in response to DNA damage.
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In this study, we have used a monoclonal antibody to characterize the behavior of hRAD51C at sites of radiation-induced DNA damage. RAD51C accumulates in nuclear foci, which identify sites of damage, as judged by the colocalization with other DNA repair proteins, RAD51 and RPA. Colocalization of RAD51C with RAD51 foci is only partial, which is consistent with overlapping but not coinciding functions for these proteins. The kinetics and dependencies of recruitment of the two proteins at break sites delineate an ordered assembly pathway (Fig. 7 F). RAD51C recruitment to break sites requires ATM and NBS1 activities involved in strand resection at DSBs as well as RPA, which decorates the ssDNA overhangs produced. In response to IR, RAD51C appears at break sites with roughly similar kinetics as RAD51 (Fig. 2 A). After release from HU-induced replication arrest, RAD51C foci appeared distinctly before RAD51, indicating that under these conditions RAD51C acts before recruitment of the recombinase. In Brca2-deficient cells, RAD51C foci form in response to radiation, whereas RAD51 fails to associate with damage sites. Therefore, we propose that, during the early stages of DNA break repair by HR, RAD51C, possibly alone or more likely in complex with other paralogues, acts as a platform for the assembly of RAD51 filaments from RAD51 monomers delivered to the DSB by BRCA2. Thereafter, although RAD51 dissociates again from the break sites as recombination reactions proceed, RAD51C persists for distinctly longer periods of time. Thus, RAD51C could act as a recruitment platform for postinvasion HR activities (i.e., Holliday junction branch migration and resolution) in the late stages of DSB repair. Of the five members of the RAD51 paralogue family, only RAD51C has been shown to be part of several subcomplexes: RAD51B–RAD51C–RAD51D–XRCC2, RAD51B–RAD51C, and RAD51C–XRCC3 (Masson et al., 2001; Sigurdsson et al., 2001; Wiese et al., 2002). It is not clear from our current experiments whether RAD51C associates with DSBs continuously throughout repair or whether there is an early and a late wave of distinct RAD51C-containing protein complex assembly at break sites. Answers to this question await the availability of higher sensitivity antibodies against other members of the RAD51 paralogue family.
A role for RAD51C in DNA damage signaling
Previous studies suggested the possibility that the early role of RAD51C in DNA repair is that of facilitating assembly of RAD51 filaments at sites of DNA damage (Takata et al., 2001; French et al., 2002; Rodrigue et al., 2006). In this study, we demonstrate that, in addition to promoting RAD51 assembly, RAD51C is required for efficient checkpoint signaling to delay cell cycle progression in response to DNA damage. This function is shared between RAD51C and RPA, and we find that both proteins assemble in coinciding foci at sites of damage, specifically during S/G2 phase of the cell cycle. RPA foci form in the absence of RAD51C, suggesting that strand resection activities and RPA association with ssDNA do not require RAD51C. Conversely, RAD51C relies on RPA for its assembly at the break. This indicates that RAD51C requires RPA to mediate its association with the break site.
RPA-coated ssDNA is generated in an ATM-dependent manner (Jazayeri et al., 2006) and facilitates recruitment of the ATR–ATR-interacting protein complex (Zou and Elledge, 2003), leading to phosphorylation of the CHK1 checkpoint effector kinase. In this study, we report that RAD51C plays a similar role in checkpoint signaling by promoting phosphorylation and thereby activation of the CHK2 kinase. This role is shared by XRCC3, which is also a member of the RAD51 paralogue family and interacting partner of RAD51C. Both ATM and NBS1 are required for CHK2 phosphorylation in vivo (Matsuoka et al., 1998; Buscemi et al., 2001; Falck et al., 2001), and ATM has the ability to directly phosphorylate CHK2 in vitro (Matsuoka et al., 2000). RAD51C is also regulated by both factors, and potential ATM phosphorylation sites have been identified (Matsuoka et al., 2007). Whether RAD51C is a target for ATM phosphorylation remains to be investigated.
It is interesting to note that RAD51C is recruited to DNA break sites only in the S/G2 phase of the cell cycle. This is consistent with CHK2 activation in response to DNA damage during G1 occurring independently of RAD51C. Indeed, in vitro reconstitution experiments suggest that the MRN complex is sufficient to promote ATM activation and CHK2 phosphorylation at paired duplex DNA ends. Such ends likely model DNA breaks in G1, which is consistent with the observation that CHK2 is phosphorylated in response to DNA damage in G1 (Matsuoka et al., 1998; Lee and Paull, 2005). In experiments using a cell-free extract system, Shiotani and Zou (2009) have demonstrated that 3' ssDNA overhangs, as generated during DNA break processing in S/G2, are inhibitory to ATM activation and CHK2 phosphorylation when compared with unprocessed ends. Therefore, it is conceivable that, in vivo during the S/G2 phases of the cell cycle, additional factors act at DNA ends to promote the generation and/or propagation of the checkpoint signal. Our results identify RAD51C as one such factor with a role in transduction of the checkpoint signal to CHK2. How RAD51C acts at DNA break sites to achieve this is an interesting question for future studies. It is possible that such a role could be direct, with RAD51C acting to recruit or mediate recruitment of canonical checkpoint proteins. Alternatively, it is possible that RAD51C plays an indirect role, being required for structural chromatin changes at the break site that allow efficient generation or propagation of the checkpoint signal. Both could be consistent with a more pronounced requirement for RAD51C at low IR doses.
An important implication of our results is that RAD51C controls RAD51 filament and HR activation at multiple levels, the earliest of which is by modulating the DNA damage signal emanating from DSBs, which halts cell cycle progression and reduces Cdk activity. In turn, this allows BRCA2 to interact with RAD51 and engage the RPA-coated ssDNA in HR reactions leading to repair (Jazayeri et al., 2006). In support of this model, we observed similar cell cycle regulation of IR-induced RAD51C, RPA, and RAD51 focus formation, all being restricted to S and G2 (Yuan et al., 2003; unpublished data).
The cell cycle arrest defect observed in RAD51C-depleted cells in response to IR could be explained by reduced CHK2 phosphorylation. CHK2 is phosphorylated in both S and G2 cells exposed to IR and controls the intra-S checkpoint (Falck et al., 2001) as well as Cdk activity required for the G2/M transition (Matsuoka et al., 1998). Consistent with RAD51C-dependent regulation of CHK2 activation, we observe both a less robust S-phase delay and an increase in the fraction of cells entering mitosis 2 h after DNA damage. RAD51C-defective cells accumulate endogenous DNA damage and enter mitosis with unrepaired DSBs, most likely as the result of defective CHK2-dependent checkpoint control at the G2/M transition. Conversely, RAD51C accumulation at sites of damage seems unaffected by the absence of CHK2. Furthermore, RAD51C function in checkpoint mediation appears independent of RAD51 filament assembly, as lack of RAD51 does not affect integrity of the CHK2 checkpoint. Together, our data support a role for RAD51C as a mediator in the early steps of DNA damage signaling (Fig. 7 F) in addition to its role in promoting HR reactions at the break sites.
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Single-cell clones were established as previously described (Bekker-Jensen et al., 2006). In brief, U2OS cell lines stably expressing GFP-RAD51C (a gift from J.-Y. Masson, Laval University Cancer Research Center, Quebec City, Quebec, Canada) or RAD51C* were generated by transfecting the expression construct alone (RAD51C*) or together with the pBabe–puro construct. After selection with 3 µg/ml puromycin for 14 d, single-cell clones were derived by serial dilutions in 96-well plates and tested for expression of the target protein.
irradiation was performed using a 137Cs source at the dose indicated. The ATM inhibitor Ku55933 (a gift from G. Smith, AstraZeneca, Manchester, England, UK; Hickson et al., 2004) was added at 10-µM final concentration for 30 min before irradiation. Caffeine (Sigma-Aldrich) was added at 5-mM final concentration 2 h before irradiation. For HU treatment, cells were incubated 8–12 h after seeding with 2 mM HU for 17 h. Then, cells were incubated with fresh medium for the indicated period of time. For the 1.5 keV ultrasoft x-ray irradiation, cells were plated on the 0.9-µm thick Mylar base of an irradiation dish. The dish was placed in contact with an irradiation mask patterned with a 1-µm thick gold grid, through which the cells were irradiated. The grid consists of a series of 9-µm wide stripes separated by 1-µm gaps, resulting in cells being irradiated with a series of 1-µm stripes at 10-µm intervals.
Immunofluorescence
For indirect immunofluorescence staining, cells were seeded onto coverslips 24 h before irradiation. When CAPAN-1 cells were used, coverslips were coated with a solution of 0.1 mg/ml fibronectin before plating the cells. After irradiation, cells were allowed to recover for the indicated times, washed in PBS, and swollen in hypotonic solution (85.5 mM NaCl and 5 mM MgCl2, adjusted to pH 7.0) for 5 min. For ultrasoft x ray–treated cells, 0.1% Triton X-100 was added to the hypotonic solution. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized by adding 0.03% SDS to the fixative, and immunostained as described previously (Tarsounas et al., 2003). For time point collection, cells were kept in PBS on ice for up to 2 h before processing. Dried coverslips were mounted on microscope slides using the ProLong Antifade kit (Invitrogen) supplemented with 1 µg/ml DAPI. Specimens were viewed with an inverted microscope (DMI6000B; Leica) and fluorescence imaging workstation equipped with an HCX Plan-Apochromat 100x NA 1.4-0.7 oil objective, and images were acquired at room temperature using a digital camera (DFC350 FX R2; Leica) using LAS-AF software (Leica). Brightness and contrast adjustments were applied to the whole image using Photoshop CS3 (Adobe).
Immunoblotting
Cells were harvested by trypsinization, washed once with cold HBSS, and resuspended in cold buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.3 M sucrose, 10% glycerol, 1 mM DTT, and 0.05% Triton X-100) supplemented with protease and phosphatase inhibitor cocktail tablets (Roche) at a concentration of 2 x 107 cells/ml. After extraction on ice for 5 min, the cell suspension was cleared by centrifugation at maximum speed in a bench-top centrifuge (model 5424; Eppendorf). The nuclear pellet was resuspended in SDS-PAGE loading buffer and sonicated. Equal amounts of protein (50–100 µg) were analyzed by gel electrophoresis followed by Western blotting. NuPAGE-Novex 10% Bis-Tris and 10% Tris-Gly gels (Invitrogen) were run according to the manufacturer's instructions. Antitubulin, anti–histone H3, or anti–histone H2AX antibody was used for a loading control.
Antibodies
Mouse monoclonal antibodies 2H11 raised against hRAD51C, 10F1 raised against human XRCC3, 3E1 raised against GFP, and TAT-1 raised against 
-tubulin (Masson et al., 2001; Tarsounas et al., 2004b) were obtained from the Cancer Research UK monoclonal antibody service. Mouse IgGs were purified from hybridoma supernatants using Gentle Ag/Ab Elution Buffer (Thermo Fisher Scientific). Rabbit polyclonal antibodies were raised against full-length RAD51 (FBE2; Barlow et al., 1997) and the 70-kD subunit of RPA (SWE34; a gift from S. West, Cancer Research UK, Hertfordshire, England, UK). Additional antibodies used were anti-H2AX (EMD); anti–histone H3 (a gift from A. Verreault, University of Montréal, Montréal, Quebec, Canada); anti–CENP-F (Abcam); anti–phospho histone H3–Ser 10 (6G3), anti–phospho CHK1–Ser 317, anti–phospho CHK2–Thr 68, and anti-CHK2 (all from Cell Signaling Technology); anti–phospho CHK2 (R&D Systems); anti-CHK2/Cds1 (Millipore); and anti-SMC1 and anti–phospho SMC1–Ser 966 (Bethyl Laboratories, Inc.). The anti–mouse RAD51C rabbit serum MTA37 was generated against the N-terminal peptide RELVGYPLSPAVRGKGKLVAAGFQTAED. The serum was affinity purified using the peptide cross-linked to Sulfo Link gel (Thermo Fisher Scientific).
RNAi
HeLa and U2OS cells were transfected using Oligofectamine (Invitrogen). In brief, 0.2–0.5 x 106 cells/well of a 6-well plate were plated 24 h before transfection. Cells were transfected with 2 µg siRNA/well twice at 7-h time intervals and processed 48 h later. siRNA duplexes were 21 base pairs with a 2-base deoxynucleotide overhang (Thermo Fisher Scientific). The sequences of RAD51C siRNAs were 5'-CACCTTCTGTTCAGCACTAGA-3' (51C siRNA; Rodrigue et al., 2006) and 5'-GAGAATGTCTCACAAATAA-3' (51C-1 siRNA); the sequences of XRCC3 siRNAs were 5'-GAATTATTGCTGCAATTAA-3' (X3 siRNA) and 5'-GCCAGATCTTCATCGAGCA-3' (X3-1 siRNA; Rodrigue et al., 2006); the sequence of RPA siRNA was 5'-AACACTCTATCCTCTTTCATGTT-3' (Zou and Elledge, 2003; McCabe et al., 2006); and the sequence of RAD51 siRNA was 5'-CTTTGGCCCACAACCCAT-3'. A GFP siRNA with the sequence 5'-GCTGACCCTGAAGTTCATCTT-3' (Tarsounas et al., 2004b) was used as a control. For expression of siRNA-resistant RAD51C (RAD51C*), the plasmid pcDNA3-RAD51C was modified by site-directed mutagenesis as previously described (Rodrigue et al., 2006).
Computational RAD51 and RAD51C foci analysis
Image analysis was performed after acquisition using the TRI2 software package, based on the Compact Hough and Radial Map algorithms (Barber et al., 2001, 2007). The algorithm was used to detect nuclei and then to count foci within the nuclei from images acquired using DAPI and Cy3 fluorescent channels.
G2/M checkpoint assay
The G2/M checkpoint assay was performed as previously described (Kolas et al., 2007). In brief, U2OS cells were transfected with control or RAD51C siRNA twice at 18-h time intervals, as described in RNAi, and, 48 h later, were exposed to 1 Gy of IR. After 2 h, cells were harvested, washed twice in PBS, and fixed in 1% paraformaldehyde at 37°C for 10 min. After permeabilization in ice-cold 90% methanol, cells were washed in PBS and stained with anti–phospho histone H3–Ser 10 antibody followed by Alexa Fluor 488–conjugated goat anti–mouse IgG secondary antibody (Invitrogen). Cells were incubated with 5 µg/ml propidium iodide and 0.25 mg/ml RNase I in PBS. At least 10,000 cells were analyzed by flow cytometry (BD Biosciences). Data were processed using CellQuest (BD Biosciences).
MEF retroviral transduction and metaphase preparation
Primary MEFs were isolated from day 13.5 embryos as previously described (Blasco et al., 1997) and cultivated in Dulbecco's minimum essential medium supplemented with antibiotics (penicillin and streptomycin) and 10% FBS. Retroviral transduction of primary MEFs was performed as previously described (Palmero and Serrano, 2001). In brief, HEK293T packaging cells were grown to 70% confluency and transfected with pRetroSuper-shRNA and pCL-Eco helper vector using a standard calcium phosphate protocol. The media was replaced 24 h after transfection. Recipient MEFs were plated and infected 24 h later with the retroviral supernatants produced by the HEK293T cells. Additional infections were performed after 24 and 36 h. 24 h after the last infection, cells were washed and incubated with fresh media containing 2 µg/ml puromycin for 48 h. Exponentially growing MEFs (obtained from A. Zelensky, Medical Research Council, Harwell, England, UK) were either collected and processed for Western blot analysis, as described in Immunoblotting, or incubated with 0.1 µg/ml colcemid (Invitrogen) for 4 h at 37°C and then fixed in methanol/acetic acid (3:1). FISH was performed as previously described (Tarsounas et al., 2004b) using 15 µg/ml Cy3-conjugated PNA[CCCTAA]3 telomeric probe (Applied Biosystems), and chromosomes were visualized with DAPI.
Online supplemental material
Fig. S1 illustrates formation of RAD51C foci in response to increasing doses of irradiation. Fig. S2 demonstrates the specificity of the RAD51C monoclonal antibody and accumulation of GFP-RAD51C foci in nuclear stripes induced by partial irradiation of cells with ultrasoft x rays. Fig. S3 shows inhibition of IR-induced RAD51C foci by caffeine. Fig. S4 examines a possible physical interaction between RAD51C and RPA. Fig. S5 shows lack of CHK2 activation in irradiated U2OS cells depleted of RAD51C using a second siRNA (RAD51C-1) as well as in RAD51C-depleted MEFs and normal CHK2 activation in irradiated U2OS cells depleted of RAD51. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200811079/DC1.
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Acknowledgments |
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This work was supported by Cancer Research UK.
Submitted: 14 November 2008
Accepted: 21 April 2009
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References |
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