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.

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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<0.05; one-tailed Welch's test). Although the repair defect is apparent and similar for the different cell systems, the absolute foci numbers in HeLa cells are higher, likely due to their higher DNA content. Error bars in panels A–C represent the s.e.m. from at least three different experiments.

HR represents the slow component of DSB repair in G2

To examine the role played by HR in G2, we first assessed the kinetics of DSB repair in Brca2-deficient HSC62 primary human fibroblasts (Howlett et al, 2002). As expected, HSC62 cells show kinetics indistinguishable from those of normal human fibroblasts in G1, but, interestingly, exhibit elevated level of unrepaired DSBs at prolonged repair times (4 h) in G2 (Figure 2A). Since the fast component of DSB repair (2 h) is unaffected in HSC62 cells both in G1 and in G2, this suggests that HR specifically represents the slow component of DSB repair in G2, which repairs ~15% of the IR-induced DSBs. Strikingly, ATM- and Artemis-deficient cells exhibit repair kinetics indistinguishable from HSC62 cells in G2 (Figure 2A and Supplementary Figure 2). We also measured DSB-repair kinetics in MEFs deficient in ATM, Artemis or Rad54. Similar to our results with primary human fibroblasts, we observed an identical repair defect in ATM^−/− and Artemis^−/− MEFs affecting the slow DSB-repair component in G1 and in G2 (Figure 2B). Rad54^−/− MEFs, in contrast, show repair kinetics indistinguishable from wild-type (WT) MEFs in G1, but a repair defect similar to ATM and Artemis in G2 (Figure 2B). Finally, we applied RNAi technology to compare deficiencies of ATM, Artemis, Brca2 or Rad51 in an isogenic background (Figure 2C). Consistent with the results using primary human fibroblasts and MEFs, HeLa cells downregulated for ATM or Artemis exhibit a repair defect in G1 and in G2, whereas cells treated with Brca2 or Rad51 siRNA show a defect only in G2, which is similar to the ATM and Artemis repair defect (Figure 2C). Significantly, the repair defects manifest only at later times (>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).

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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).

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.

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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.

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.

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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.

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 γH2AX foci counting in combination with epistasis analysis. Addition of the ATM inhibitor, KU55933, to WT, Artemis- and Brca2-deficient cells confirmed that ATM and Artemis function in the same repair pathway in G1 and G2, which is epistatic with Brca2 in G2 (Figure 6A). Importantly, KU55933 increased the level of unrepaired DSBs in XLF-deficient primary human fibroblasts in G2 but not in G1 (Figure 6A). Indeed, the elevation in the level of unrepaired DSBs conferred by KU55933 in G2 appears to be the same in XLF-deficient cells and in WT cells. Hence, in G2, there is additivity for the loss of XLF and ATM instead of epistasis. Further, KU55933 did not affect foci levels in Rad54^−/− MEFs in G2, demonstrating epistasis between ATM and Rad54 in G2 (Figure 6B). In contrast, KU55933 increased foci levels in Lig4^−/− MEFs in G2 (Figure 6B). XRCC2^−/− MEFs provided results similar to Rad54^−/− MEFs (Supplementary Figure 6). We also applied RNAi technology to exclude the possibility that KU55933 has off-target effects (Figure 6C). HeLa cells downregulated in the combinations ATM/Brca2, ATM/Rad51, Artemis/Brca2 or Artemis/Rad51 show a defect similar to that observed after downregulation of the individual factors (Figure 6C). Taken together, this confirms our finding that ATM and Artemis function in the same pathway as Brca2, Rad51 and Rad54 for repairing radiation-induced DSBs in G2.

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Figure 6

(A) γH2AX foci in primary human fibroblasts in the presence or absence of the ATM inhibitor, KU55933 (designated ATMi in the figure). The insets magnify the 8-h data for WT, Brca2-; Artemis- and ATM-deficient cells treated with KU55933. (B) γH2AX foci analysis in MEFs in the presence or absence of KU55933. Statistical analysis was performed at the 6- and 8-h time points, and revealed that XLF- and Lig4-deficient cells treated with KU55933 exhibit significantly elevated foci levels compared with untreated XLF- and Lig4-deficient cells in G2 but not in G1 (P<0.05; one-tailed Welch's test). (C) γH2AX foci analysis in siRNA-treated HeLa cells. Samples were evaluated in a blinded manner. Statistical analysis was performed and revealed that cells depleted for Artemis, ATM, Control/Artemis, Rad51/Artemis, Rad51/ATM, Brca2/Artemis and Brca2/ATM in G1 and G2, and Rad51-, Brca2- and Rad51/Brca2-depleted cells in G2 exhibit significantly elevated foci levels compared with control siRNA- and mock-treated cells (P<0.05; one-tailed Welch's test). Samples designated ‘mock' were treated with transfection reagents, but no siRNA was added. The experiments for panels A–C were carried out as for Figure 2. Error bars represent the s.e.m. from at least three different experiments. (D) Analysis of G2 PCC chromosomal breaks in calyculin A-treated primary human fibroblasts in the presence of aphidicolin and in the presence or absence of KU55933. Breaks in un-irradiated samples were less than 0.2 and were subtracted from the breaks in the irradiated samples. Error bars represent the s.e.m. from at least 100 cells. Statistical analysis revealed that Brca2- and Artemis-deficient cells with and without KU55933 exhibit significantly more PCC breaks than WT cells without KU55933 at 4 and 6 h (P<0.05; one-tailed Welch's test).

As an alternative approach to examine DSB repair in primary human fibroblasts, we induced premature chromosome condensation (PCC) in G2-phase cells using the phosphatase inhibitor calyculin A. G2 cells are readily distinguished from mitotic cells and allow analysis of PCC breaks (Asakawa and Gotoh, 1997). Enumeration of PCC breaks in G2-phase chromosomes confirmed a repair defect in Artemis- and Brca2-deficient cells at 4 and 6 h, which is not observable at 1 h following 1-Gy IR treatment, a phenotype consistent with the analysis of γH2AX foci (Figure 6D). ATM inhibition in WT, Artemis- and Brca2-deficient cells confirmed that ATM, Artemis and Brca2 operate in the same pathway of DSB repair in G2 (Figure 6D).

Taken together, our epistasis studies applying γH2AX foci analysis and PCC technology show that ATM and Artemis function in the same DSB repair pathway as Brca2, Rad51 and Rad54 in G2, and, hence, confirm the notion that ATM and Artemis have a role in promoting HR of IR-induced DSBs in G2.

Artemis-dependent DSB repair in G2 does not require DNA-PK activity

Artemis' role in V(D)J recombination and IR-induced DSB repair in G1 has been shown to require DNA-PK activity (Riballo et al, 2004; Goodarzi et al, 2006; van Gent and van der Burg, 2007). Given its involvement in HR and the finding that it functions independently of XLF and Lig4 in G2, we next tested whether Artemis functions in G2 independently of DNA-PK activity. Treatment of WT primary human fibroblasts with the DNA-PK inhibitor, NU7026, resulted in substantially impaired DSB-repair kinetics in both G1 and G2, consistent with evidence that NHEJ has a major role in both cell-cycle phases. In G1, NU7026-treated, Artemis-deficient cells exhibit the same defect as NU7026-treated WT cells (Figure 7A). Thus, loss of Artemis does not confer an additional repair defect in a DNA-PK-deficient background, consistent with Artemis' role requiring DNA-PK (Riballo et al, 2004). However, in G2, NU7026-treated, Artemis-deficient cells show a repair defect greater than that observed in NU7026-treated WT cells (Figure 7A). Indeed, the elevation in the level of unrepaired DSBs in DNA-PKcs-inhibited, Artemis-deficient cells (relative to WT cells) appears to be the sum of the elevation in DSBs in cells deficient for Artemis and in cells inhibited for DNA-PKcs. Hence, in G2, there is additivity for the loss of Artemis and DNA-PKcs instead of epistasis.

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Figure 7

(A) γH2AX foci analysis in primary human fibroblasts in the presence or absence of the DNA-PK inhibitor, NU7026 (designated DNA-PKi in the figure). The analysis was carried out as for Figure 2A. Statistical analysis was performed at the 6- and 8-h time points, and revealed that Artemis- and Brca2-deficient cells with NU7026 exhibit significantly elevated foci levels compared with WT cells with NU7026 in G2 but not in G1 (P<0.05; one-tailed Welch's test). (B) γH2AX foci analysis at prolonged times after IR. Using an approach, which circumvents aphidicolin treatment, cells were pulse-labelled with BrdU for 1 h and irradiated with 9 Gy 4 h after labelling (when in G2). Due to the higher dose, the majority of irradiated G2 cells remained in G2 for at least 96 h (Supplementary Figure 7A). Foci were analysed in BrdU-positive cells and taken to represent G2 cells. The few BrdU-positive cells that had progressed from G2 into G1 during repair incubation were excluded from the analysis based on their lower DAPI signal compared with that of G2 cells. Data for G0 cells were obtained in parallel experiments from the analysis of confluent cultures. Results similar to G0 were obtained for G1 from the analysis of BrdU-negative cells (representing mainly G1 cells) in the same samples that were analysed for the G2 data (Supplementary Figure 7B). The continuing activity of NU7026 over 96 h was confirmed in control experiments (Supplementary Figure 7C). Error bars represent the s.e.m. from the analysis of at least three different experiments.

We have previously shown that Artemis-independent DSBs in G1 can be repaired without DNA-PKcs, although at a reduced rate, and that the level of unrepaired DSBs in a DNA-PKcs-defective background reaches the level of unrepaired DSBs in Artemis-deficient cells at prolonged repair times (Riballo et al, 2004). Hence, we concluded that DNA-PKcs has an essential role in Artemis-dependent NHEJ, but a non-essential, facilitating role for Artemis-independent DSBs (Löbrich and Jeggo, 2005). Here we show that the level of unrepaired DSBs in DNA-PKcs-inhibited G1 cells is similar to that of Artemis-deficient G1 cells at repair times 24 h (Figure 7B and Supplementary Figure 7A–C). Moreover, Artemis-deficient G2 cells exhibit twice the level of unrejoined DSBs compared with Artemis-deficient G1 cells, consistent with an essential role of Artemis for the same subset of DSBs in G1 and in G2. However, DNA-PKcs-inhibited G2 cells have a much lower level of unrejoined DSBs than Artemis-deficient G2 cells at repair times 24 h, and show only a marginal elevation compared with WT cells (Figure 7B). Strikingly, at repair times 48 h, DNA-PKcs-inhibited cells display fewer unrejoined DSBs in G2 than in G1 (Figure 7B). This strongly suggests that in G2 there is no absolute requirement for DNA-PK activity for DSB repair (although NHEJ in G2 is facilitated by DNA-PKcs to the same degree as in G1) and that the essential role of Artemis in G2 for a subset of IR-induced DSBs is independent of DNA-PKcs. Taken together, our epistasis studies using DNA-PKcs inhibitors support the notion that the role of ATM and Artemis in G2 is independent of DNA-PK-dependent NHEJ.

Artemis endonuclease activity promotes DSB repair by HR

We have previously shown that Artemis endonuclease activity is important for the slow component of DSB repair by NHEJ in G1 (Riballo et al, 2004). Here, we analysed G2-phase, Artemis-deficient primary human fibroblasts that were efficiently transfected with Artemis expression constructs (Figure 8). Transfection with WT Artemis complemented the G2 DSB-repair defect of Artemis-deficient cells. Strikingly, transfection with the endonuclease-deficient Artemis construct (D37N) did not restore normal DSB repair (Figure 8). Furthermore, expression of WT Artemis, but not D37N Artemis, restored the Rad51 foci level of Artemis-deficient fibroblasts. We also overexpressed Artemis in control cells and observed that D37N-mutant Artemis, but not WT Artemis, increased the γH2AX foci level and decreased the Rad51 foci level (Figure 8). This provides strong evidence that Artemis promotes the early steps of HR as an endonuclease, potentially during the processing step, which is necessary to enable resection of radiation-induced DSBs.

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Figure 8

Artemis endonuclease activity promotes IR-induced HR in G2. The DSB-repair defect of Artemis-deficient cells (CJ179) is corrected with WT Artemis but not with an endonuclease-deficient Artemis construct (D37N), and overexpression of D37N confers a repair defect to HSF1 control cells. Analysis of γH2AX and Rad51 foci was conducted in cells positive for c-myc, that is, only cells efficiently transfected were included in the analysis. The image on the left shows two G2-phase, Artemis-deficient cells transfected with the WT Artemis construct at 8 h following 2-Gy irradiation. One of the two cells shows efficient Artemis expression (c-myc signal) and lower foci numbers. Error bars represent the s.e.m. from three different experiments. Statistical analysis revealed that Artemis-deficient cells with or without complementation of the Artemis D37N construct exhibit significantly elevated γH2AX foci levels (at 6 and 8 h) and significantly reduced Rad51 foci levels (at 2 h) compared with Artemis-deficient cells complemented with the Artemis WT construct (P<0.05; one-tailed Welch's test). Control cells overexpressing D37N-mutant Artemis exhibit significantly elevated γH2AX foci levels (at 6 and 8 h) and significantly reduced Rad51 foci levels (at 2 h) compared with control cells with or without overexpression of Artemis WT (P<0.05; one-tailed Welch's test). See Supplementary data for further information.

Immunofluorescence

Cells grown on coverslips were fixed in 100% methanol (−20°C) for 30 min, permeabilized in acetone (−20°C) for 1 min and washed 3 × 10 min in PBS/1% FCS. Samples were incubated with primary antibodies (monoclonal or polyclonal anti-γH2AX antibody 1:200, Upstate; polyclonal anti-CENP-F antibody 1:200, Santa Cruz; polyclonal anti-phosphoH3 (pH3) antibody (Ser10) 1:200, Upstate; monoclonal anti-BrdU antibody 1:200, Becton Dickinson; polyclonal anti-RPA antibody 1:100, Calbiochem and anti -Rad51 antibody 1:100, Santa Cruz) in PBS/1% FCS for 1 h at room temperature, washed in PBS/1% FCS for 3 × 10 min and incubated with Alexa Fluor 488-, Alexa Fluor 546- or Alexa Fluor 594-conjugated secondary antibodies (MoBiTec, 1:500) for 1 h at room temperature. Cells were washed in PBS for 4 × 10 min and mounted using Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). In a single experiment, cell counting was performed until at least 40 cells and 40 foci were registered per sample. For data points that were derived from a single experiment, the error bars represent the s.e.m. from the analysis of all cells. For data points that were derived from more than one experiment, the error bars represents the s.e.m. between the independent experiments, which is typically higher than the s.e.m. from all analysed cells. Statistical analysis using the Student's t-test was performed at critical time points to evaluate the significance of differences in foci levels.

We thank Dr G Smith for providing the ATM inhibitor KU55933. The ML laboratory is supported by the Deutsche Forschungsgemeinschaft (Grant LO 677/4-1/2), the Bundesministerium für Bildung und Forschung via the Forschungszentrum Karlsruhe (Grants 02S8135 and 02S8355) and the Wilhelm Sander-Stiftung (2003.114.1/3). The PAJ laboratory is supported by the Medical Research Council, the Association for International Cancer Research, the Leukaemia Research Fund, the Department of Health and EU grant (FIGH-CT-200200207) (DNA repair). Both laboratories are supported by EU Grant FI6R-CT-2003-508842 (RiscRad).