ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2
2009; Springer Nature; Volume: 28; Issue: 21 Linguagem: Inglês
10.1038/emboj.2009.276
ISSN1460-2075
AutoresAndrea Beucher, Julie Birraux, Leopoldine Tchouandong, Olivia Barton, Atsushi Shibata, Sandro Conrad, Aaron A. Goodarzi, Andrea Krempler, Penny A. Jeggo, Markus Löbrich,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoArticle24 September 2009Open Access ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2 Andrea Beucher Andrea Beucher Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Julie Birraux Julie Birraux Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Leopoldine Tchouandong Leopoldine Tchouandong Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Olivia Barton Olivia Barton Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Atsushi Shibata Atsushi Shibata Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Sandro Conrad Sandro Conrad Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Aaron A Goodarzi Aaron A Goodarzi Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Andrea Krempler Andrea Krempler Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Penny A Jeggo Corresponding Author Penny A Jeggo Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Markus Löbrich Corresponding Author Markus Löbrich Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Andrea Beucher Andrea Beucher Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Julie Birraux Julie Birraux Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Leopoldine Tchouandong Leopoldine Tchouandong Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Olivia Barton Olivia Barton Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Atsushi Shibata Atsushi Shibata Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Sandro Conrad Sandro Conrad Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Aaron A Goodarzi Aaron A Goodarzi Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Andrea Krempler Andrea Krempler Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Penny A Jeggo Corresponding Author Penny A Jeggo Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Markus Löbrich Corresponding Author Markus Löbrich Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany Search for more papers by this author Author Information Andrea Beucher1,‡, Julie Birraux2,‡, Leopoldine Tchouandong1,‡, Olivia Barton1, Atsushi Shibata2, Sandro Conrad1, Aaron A Goodarzi2, Andrea Krempler1, Penny A Jeggo 2 and Markus Löbrich 1 1Darmstadt University of Technology, Radiation Biology and DNA Repair, Darmstadt, Germany 2Genome Damage and Stability Centre, University of Sussex, East Sussex, UK ‡These authors contributed equally to this work *Corresponding authors: Genome Damage and Stability Centre, University of Sussex, East Sussex BN1 9RQ, UK. Tel.: +44 1273 678482; Fax: +44 1273 678121; E-mail: [email protected] University of Technology, Radiation Biology and DNA Repair, Darmstadt 64287, Germany. Tel.: +49 6151 167460; Fax: +49 6151 167462; E-mail: [email protected] The EMBO Journal (2009)28:3413-3427https://doi.org/10.1038/emboj.2009.276 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of ∼15% of X- or γ-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ. Introduction Homologous recombination (HR) and non-homologous end joining (NHEJ) represent two conceptually different pathways for repairing DNA double-strand breaks (DSBs), the lesion at the heart of many physiological and pathophysiological processes in mammalian cells. HR uses homologous sequences on the sister chromatid as a template to restore the genomic integrity upon DSB induction and involves genes of the Rad52 epistasis group (Thompson and Schild, 2002; West, 2003; Wyman and Kanaar, 2006; Thorslund and West, 2007). In contrast, NHEJ repairs DSBs without requiring sequence homology and involves the core components DNA-PKcs/Ku70/Ku80 and DNA ligase IV/XRCC4/XLF (Nussenzweig and Nussenzweig, 2007; van Gent and van der Burg, 2007). HR has a major role in repairing one-sided DSBs that arise at collapsed replication forks; it can also repair two-ended DSBs in G2-phase (Trenz et al, 2006; Hanada et al, 2007; Roseaulin et al, 2008). Perhaps the strongest evidence for this latter function in mammalian cells is provided by studies involving site-specific introduction of DSBs, frequently using an I-SceI restriction site on plasmids or integrated model substrates (Liang et al, 1996; Taghian and Nickoloff, 1997; Moynahan et al, 1999; Johnson and Jasin, 2000; Allen et al, 2002). Indirect evidence for a role of HR in repairing ionizing radiation (IR)-induced DSBs is provided by the finding that HR-deficient cells exhibit increased sensitivity in the S and G2-phases of the cell cycle. However, mutants of the NHEJ pathway are exquisitely sensitive throughout the cell cycle and exhibit severe deficiency for repairing IR-induced DSBs (Rothkamm et al, 2003; Hinz et al, 2005). Here we aimed to address the role of HR in the repair of radiation-induced DSBs that arise in G2-phase. In addition to the core components of NHEJ, several processing factors have been identified (van Gent and van der Burg, 2007; Lieber, 2008; Weterings and Chen, 2008). ATM, which is defective in the disorder, ataxia telangiectasia (A-T), and Artemis, a nuclease involved in hairpin opening during V(D)J recombination (Pannicke et al, 2004; Soulas-Sprauel et al, 2007; van der Burg et al, 2007), are required in non-cycling G0 cells for the repair of a subset of radiation-induced DSBs by NHEJ (Riballo et al, 2004; Wang et al, 2005; Darroudi et al, 2007). This subset represents the slow component of DSB repair, which are those DSBs located within or close to regions of heterochromatin (Goodarzi et al, 2008). Recently, we reported that ATM and Artemis also function to repair a similar sized fraction of IR-induced DSBs (approximately 15%) in G2 (Deckbar et al, 2007; Löbrich and Jeggo, 2007). To analyse DSB repair in G2-phase, we irradiate asynchronous cell populations with physiological doses that allow survival, and apply immunofluorescence analysis to study DSB repair by enumerating γH2AX foci at defined times post-IR. Using centromere protein F (CENP-F) staining to identify G2 cells and aphidicolin to prevent cells in S-phase from progressing into G2 during repair incubation, we study DSBs that are generated and processed specifically in G2. We have developed a technology based on the incorporation of BrdU during repair synthesis that allows us to specifically study HR events after irradiation in G2 and also monitor the formation of RPA and Rad51 foci to assess the process of HR. Finally, we designed an approach to detect HR events based on the appearance of sister-chromatid exchanges (SCEs) that arise after IR in G2. Collectively, our findings show that NHEJ represents the major DSB-repair pathway in G2, with HR only being essential for the repair of a minor subset (∼15%) of IR-induced DSBs. We show that DSB repair by HR in G2 involves ATM and Artemis endonuclease function and provide evidence that the DSBs repaired by HR are located at the heterochromatin. Thus, HR is specifically required to repair the slow DSB-repair component after exposure to X- or γ-rays. Importantly, Artemis is not a specific NHEJ factor and Artemis and ATM are involved in HR or NHEJ depending on the cell-cycle phase. Results DSB-repair measurements during the mammalian cell cycle To investigate the contribution of NHEJ and HR in different cell-cycle phases, we analysed asynchronously growing cells to avoid potential introduction of DSBs during synchronization. For human fibroblasts, we used pan-nuclear CENP-F staining to identify G2 cells (Liao et al, 1995; Kao et al, 2001) and added aphidicolin to prevent irradiated S-phase cells progressing into G2 during analysis (Figure 1A). Aphidicolin caused pronounced pan-nuclear H2AX phosphorylation in S-phase cells, likely due to DSBs resulting from collapsed replication forks (Figure 1A). These S-phase cells, as well as mitotic cells, identified by their condensed chromatin and CENP-F staining were excluded from analysis. Flow cytometry (FACs) analysis demonstrated that up to 8 h post-IR the majority of irradiated G2 cells remain in G2 and that S-phase cells do not progress into G2 (Supplementary Figure 1A), providing sufficient time to detect the repair defect in ATM- and Artemis-deficient cells, which is measurable at >4 h post-IR (Riballo et al, 2004). To identify cell-cycle phases in mouse embryonic fibroblasts (MEFs), phosphoH3 was used instead of CENP-F (Supplementary Figure 1B). We used γH2AX foci analysis to monitor DSB repair. Figure 1.(A) Identification of cell-cycle phases in human fibroblasts (HSF1). Cells were scanned under the microscope and the γH2AX signal was plotted against the DAPI signal. S-phase cells exhibited an intermediate DAPI signal, a weak CENP-F signal and a high pan-nuclear γH2AX signal due to aphidicolin treatment. G1- and G2-phase cells were distinguished from S-phase cells by their dotted instead of a pan-nuclear γH2AX signal. G1 and G2 cells were distinguished from each other either by DAPI content (low in G1 versus high in G2) or by CENP-F staining (absent in G1 versus strong pan-nuclear in G2). Mitotic cells exhibited CENP-F staining and condensed chromatin (Deckbar et al, 2007), and were excluded from the analysis. (B) DSB repair in G1- and G2-phase HSF1 cells is unaffected by aphidicolin treatment and aphidicolin itself (designated 'control' in the figure) does not induce γH2AX foci. (C) γH2AX foci formation in HSF1 cells at 15 min post IR is linear with dose in G1 and G2 up to approximately 80 foci per cell. G2 cells exhibit about twice the number of DSBs as G1 cells, consistent with their two-fold higher DNA content (NB: The number of DSBs observed in G2 phase at later times (e.g. 8 h) is routinely higher than twice the number in G1-phase since the slow component of DSB repair in G2 is slower than in G1). Error bars in panels B and C represent the s.e.m. from analysis of at least 40 cells. Download figure Download PowerPoint Enumeration of γH2AX foci in CENP-F-positive primary human fibroblasts following 2-Gy X-irradiation provided DSB-repair kinetics in G2 (Figure 1B). Enumeration of γH2AX foci in CENP-F-negative cells, which were also negative for the pan-nuclear aphidicolin-induced γH2AX signal, allowed the analysis of repair in G1 (Figure 1B). We observed similar kinetics and magnitude of repair in G1 and G2, which was also similar to that previously observed in G0 cells (Riballo et al, 2004). Aphidicolin treatment did not affect the repair capacity and did not form γH2AX foci in G1 or G2 (Figure 1B). Initial γH2AX foci increased linearly with dose up to 80–90 foci per cell (Figure 1C and Supplementary Figure 1C). Foci numbers correlated with DNA content, being twice as high in G2 compared with G1 (Figure 1C). Since γH2AX foci in G1 are lost in a manner entirely dependent on NHEJ factors and represent DSBs (Riballo et al, 2004), the two-fold higher numbers in G2 strongly suggest that γH2AX foci in G2 also represent DSBs. To further substantiate this contention, we treated control and ATR-deficient cells with the specific ATM and DNA-PK inhibitors, KU55933 and NU7026. IR-induced γH2AX foci formation was identical between control and ATR-deficient cells and was abolished by joint KU55933/NU7026 treatment, demonstrating that ATM and DNA-PKcs, but not ATR, contribute to foci formation in G1 and G2 (Supplementary Figure 1D). NHEJ represents the major repair pathway for IR-induced DSBs in G1 and in G2 Our previous studies utilizing CHO cells suggested that NHEJ is an important repair pathway throughout the mammalian cell cycle (Rothkamm et al, 2003). To substantiate this notion, we investigated mutants with deficiencies in XLF and DNA ligase IV (Lig4), two prominent NHEJ factors (Ahnesorg et al, 2006; Buck et al, 2006; Li et al, 2008). XLF-deficient primary human fibroblasts exhibit a pronounced repair defect in G1 and in G2, with the majority of DSBs remaining unrepaired until 8 h post-IR (Figure 2A). Similar results were obtained with Lig4-defective MEFs (Figure 2B). These observations support the model that NHEJ represents the major pathway for repairing IR-induced DSBs in G1 and in G2. Figure 2.(A) γH2AX foci analysis in primary human fibroblasts. Background foci numbers in primary human fibroblasts were about 2 in G2 and 0.2 in G1, and were subtracted from the foci numbers in the irradiated samples. (B) γH2AX foci analysis in MEFs. Background foci numbers in MEFs were about 2–4 in G2 and 0.5–2 in G1, and were subtracted from the foci numbers in the irradiated samples. The insets magnify the 8-h data for WT, Brca2-; Rad54-, Artemis- and ATM-deficient cells. Statistical analysis was performed at the 6- and 8-h time points, and revealed that Artemis- and ATM-deficient cells in G1 and G2, and Brca2- and Rad54-deficient cells in G2 exhibit significantly elevated foci levels compared with WT cells (P<0.05; one-tailed Welch's test). (C) γH2AX foci analysis in siRNA-treated HeLa cells analysed 48 h after transfection. Efficient knockdown to protein levels <20% was confirmed for all tested siRNAs by Western blotting. Background foci numbers in HeLa cells were about 2–4 in G2 and 0.5–2 in G1, and were subtracted from the foci numbers in the irradiated samples. The number of induced foci measured at 15 min post-IR was similar for all siRNA conditions (data not shown). Samples were evaluated in a blinded manner. Statistical analysis was performed and revealed that cells depleted for Artemis or ATM in G1 and G2, and for Brca2 and Rad51 in G2 exhibit significantly elevated foci levels compared with control siRNA-treated cells at the 8- and 10-h time points (P 2 h) confirming that they affect the slow component of DSB repair (Figure 2C). Collectively, these results show that HR represents the slow component of DSB repair in G2, and raise the intriguing possibility that ATM and Artemis promote HR during G2. Artemis- and ATM-deficient cells show a defect in IR-induced repair synthesis during HR in G2 Since HR repairs only ∼15% of the IR-induced DSBs in G2, and since γH2AX foci loss monitors all repair events, that is, NHEJ and HR, we sought alternative techniques, which would more specifically measure HR of IR-induced DSBs in G2. We developed a technique, which is based on microscopic detection of BrdU that is incorporated into DNA during repair by HR when extensive DNA regions are synthesized (Figure 3). In this assay, BrdU is added during repair, that is, after irradiation, and the DNA is denatured for analysis. Aphidicolin was added and CENP-F was used to discriminate between G1, S and G2 cells, similar to our cell-cycle-specific approach for measuring γH2AX foci. Distinct BrdU foci after irradiation were observed only in G2 cells exhibiting pronounced CENP-F staining and high (4N) DNA content. S-phase cells with faint CENP-F staining and intermediate DNA content showed pronounced pan-nuclear BrdU signal and were excluded from analysis. The pronounced BrdU signal of S-phase cells in the presence of aphidicolin is consistent with the pronounced pan-nuclear γH2AX signal of these cells (Figure 1A), and may suggest that DSBs arising due to aphidicolin treatment are repaired by HR. Importantly, G1 cells negative for CENP-F and exhibiting low DNA content did not show any BrdU incorporation, demonstrating that any DNA synthesis during NHEJ is undetectable by this technique (Figure 3). The number of HR sites monitored with this assay in G2 cells increases with repair time and inversely correlates with the slow component of loss of γH2AX foci (HeLa cells depleted for Brca2 or Rad51 exhibit ∼7 more γH2AX foci than control cells at 8 h following 2-Gy irradiation treatment, while the BrdU incorporation assay monitors ∼14 BrdU sites at 8 h following 4-Gy irradiation treatment; compare Figure 3 with Figure 2C). The specificity of the assay is further shown by its strict requirement for Rad51 and Brca2. Moreover, depletion of Ku80 or Lig4 does not substantially affect BrdU foci levels (Figure 3 and Supplementary Figure 3). Significantly, Artemis and ATM depletion leads to complete lack of HR sites, providing strong evidence that Artemis and ATM promote HR of IR-induced DSBs in G2 (Figure 3). Figure 3.HR repair sites were visualized by incorporation of BrdU during repair synthesis. HeLa cells were irradiated with 4 Gy and BrdU was added for the entire repair time—BrdU foci were observed after denaturing conditions in G2- but not in G1-phase cells. S-phase cells were identified by pan-nuclear γH2AX or BrdU staining and excluded from the analysis. The image on the left shows control cells analysed 8 h following 4-Gy irradiation treatment. Efficient knockdown to protein levels <25% was confirmed by Western blotting and γH2AX foci analysis (Supplementary Figure 3). Error bars represent the s.e.m. from the analysis of three different experiments (two experiments for Ku80 and Lig4 siRNA). Download figure Download PowerPoint ATM and Artemis are required for IR-induced SCEs in G2 We sought further evidence for a role of ATM and Artemis in promoting G2 HR and studied the formation of SCEs as a well-established marker for HR events (Sonoda et al, 1999 and Figure 4). We grew HeLa cells in BrdU-containing medium for two cell cycles, added aphidicolin immediately before irradiation with 2 Gy to exclude the S-phase cells from analysis and collected metaphases up to 12 h post-IR. Pilot experiments had shown that G2 cells irradiated with 2 Gy enter mitosis between 8 and 12 h after irradiation (data not shown and Deckbar et al, 2007). Hence, this procedure monitors SCE formation arising from HR in G2-phase. We observed ∼7 SCEs per cell in un-irradiated cells, which increased to ∼14 SCEs per cell after IR treatment (Figure 4). Downregulation of Rad51 or Brca2 by siRNA led to significant reduction in the spontaneous SCE level and completely abolished the formation of radiation-induced SCEs (Figure 4). Spontaneous SCEs are likely produced during the preceding cell cycles when siRNA downregulation is ongoing but not yet optimal. Thus, the reduction observed after Rad51 or Brca2 depletion probably represents an underestimation. In contrast to the effects of Rad51 and Brca2, neither spontaneous nor IR-induced SCE level is significantly affected by depleting Ku80 or Lig4. Most importantly, downregulation of Artemis or ATM does not substantially affect the spontaneous SCE level, but nearly completely abolishes the increase observed after irradiation (Figure 4). Hence, Artemis and ATM play a major role in promoting HR of IR-induced DSBs in G2, but are unlikely to represent core components of the HR machinery. Figure 4.SCEs are detected in G2-irradiated HeLa cells. Cells were grown for two cell cycles (48 h) in BrdU-containing medium and aphidicolin was added immediately prior to irradiation with 2 Gy. Colcemid was then added at 8 h post-IR and the samples were harvested at 12 h post-IR. Irradiation was performed 48 h after siRNA transfection. Samples designated 'mock' were treated with transfection reagents, but no siRNA was added. SCE numbers were normalized to 70 chromosomes to account for the variability in chromosome number between metaphases. At least 100 metaphases from at least three different experiments were analysed. Error bars represent the s.e.m. from all analysed metaphases. Download figure Download PowerPoint We also examined whether Artemis and ATM are involved in HR using a plasmid-based I-SceI system (Supplementary Figure 4). We measured the frequency of reconstitution of a GFP reporter gene within a chromosomally integrated plasmid substrate in transformed human fibroblasts with or without silencing of Artemis, ATR or Brca2 (Supplementary Figure 4A), as previously described (Pierce et al, 1999). Transient expression of the I-SceI endonuclease generates a DSB, which can be repaired by gene conversion, yielding a functional GFP gene whose expression can be assessed by flow cytometry (Supplementary Figure 4B). Consistent with previous studies (Moynahan et al, 2001; Wang et al, 2004), we observed significant reduction in I-SceI-induced HR after depletion of Brca2 or ATR, but failed to observe any effect in cells depleted for Artemis (Supplementary Figure 4C). Moreover, there was only a small impact (approximately one-third) caused by inhibiting ATM (Supplementary Figure 4C). The ATR dependency perhaps suggests that this assay preferentially monitors HR events occurring during S-phase. In any case, the lack of a pronounced dependency on ATM or Artemis confirms that these factors are not core HR enzymatic components. RPA, Rad51 and ssDNA foci formation is compromised in ATM- and Artemis-deficient cells Our approaches using quantification of BrdU incorporation during HR, as well as assessment of SCE levels in G2-irradiated cells, provided strong evidence that ATM and Artemis are defective in a pathway of HR. To examine at which point the proteins might act and to gain further evidence for impaired HR, we examined the intermediates in the reaction (Figure 5). RPA and Rad51 form IR-induced foci, which have been suggested to represent sites of resected DSBs (Miyazaki et al, 2004; Bekker-Jensen et al, 2006; Sartori et al, 2007). Consistent with this, we observed that Rad51 foci are restricted to S- and G2-phase cells, and examined foci specifically in G2 cells. RPA and Rad51 foci in G2 form linearly with dose up to ∼6 Gy (Supplementary Figure 5A–C). Time-course analysis revealed that the maximum number is formed at 2–3 h post-IR, which then decreases. The rate of decrease parallels the slow component of DSB repair. The maximum number of 15–20 foci observed after a dose of 2 Gy is consistent with our notion that approximately 15–20% of the IR-induced DSBs are repaired by HR in G2 (based on an induction rate of 50 DSBs per Gy per G2 cell; Kegel et al, 2007), and our finding that NHEJ repairs the majority of DSBs in G1 and G2. Figure 5.ssDNA formation is compromised in ATM/Artemis-deficient cells. (A) Analysis of RPA and Rad51 foci formation in primary human fibroblasts. (B) Analysis of Rad51 foci formation in siRNA-treated HeLa cells. (C) Analysis of ssDNA by measuring BrdU foci in siRNA-treated HeLa cells. Cells were labelled with BrdU for 24 h prior to irradiation and foci were observed after non-denaturing conditions. Error bars represent the s.e.m. from the analysis of at least three different experiments. Download figure Download PowerPoint RPA and Rad51 foci formation is significantly compromised in primary human A-T fibroblasts (Figure 5A), consistent with previous observations (Morrison et al, 2000; Yuan et al, 2003; Shrivastav et al, 2009). Strikingly, Artemis-deficient fibroblasts show a defect in RPA and Rad51 foci formation similar to that of A-T cells (Figure 5A), substantiating our conclusion that Artemis and ATM promote HR in G2. We also investigated Brca2-deficient fibroblasts and observed, in contrast to ATM- and Artemis-deficient cells, normal formation of RPA foci. However, RPA foci failed to disappear with time in Brca2-deficient cells, remaining at ∼20 foci per cell (after 2 Gy IR) for up to 8 h (Figure 5A). At this time, the level of RPA foci is similar to that of γH2AX foci (see Figure 2A), suggesting that all unrepaired DSBs in Brca2-deficient cells exhibit unresolved RPA foci. Consistent with the requirement of Brca2 for Rad51 foci formation (Yuan et al, 1999; Esashi et al, 2007), we failed to observe Rad51 foci in Brca2-deficient cells. Moreover, HeLa cells downregulated for CtIP, a newly identified protein that promotes end resection during HR (Sartori et al, 2007; Huertas et al, 2008), show a severe defect in Rad51 and RPA foci formation (Figure 5B and data not shown). Finally, we developed a BrdU staining technique to detect the presence of single-stranded DNA (ssDNA) intermediates in G2-phase cells, which we considered would be representative of HR rather than NHEJ (Figure 5C). In this assay, cells are prelabelled with BrdU for 24 h and BrdU foci are detected under non-denaturing conditions. Importantly, this assay directly measures the presence of ssDNA without relying on the detection of recruited or modified repair factors (such as RPA or Rad51). We observed BrdU foci in G2- but not in G1-phase cells, which increase up to 2 h and then decrease with kinetics similar to the loss of RPA or Rad51 foci. We observed persistent ssDNA intermediates in Rad51-depleted cells, consistent with the notion that ssDNA arises but HR does not proceed without Rad51. Strikingly, HeLa cells downregulated for ATM or Artemis show substantially reduced ssDNA formation, verifying their role in promoting end resection during IR-induced HR in G2 (Figure 5C). Taken together, the formation and loss of RPA and Rad51 foci correlates with the subset of DSBs that is repaired by HR. Moreover, Rad51 foci do not form in cells lacking Brca2, CtIP or Rad51, substantiating the specificity of the assay. Strikingly, RPA and Rad51 foci, as well as formation of ssDNA, are considerably reduced in ATM- and Artemis-deficient cells. Epistasis analysis confirms that ATM and Artemis function in the same pathway as Brca2, Rad51 and Rad54 for repairing radiation-induced DSBs in G2 We next wished to confirm our finding that ATM and Artemis promote HR in G2 by γH
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