The G₂-phase Decatenation Checkpoint Is Defective in Werner Syndrome Cells^{1, 2}

To ensure accurate transmission of genetic information from one cell to its daughters, cells have evolved surveillance mechanisms that monitor chromosome structure and coordinate cell cycle progression with DNA repair: the cell cycle checkpoints (1). Major cell cycle checkpoints exist at G₁-S, S, and G₂-M of the cell cycle (2, 3), but anaphase transitions and the catenation status of the chromosomes are also monitored by checkpoint mechanisms (4).

The checkpoint sensitive to the catenation status of the chromosomes is called decatenation checkpoint and can be activated during the G₂ phase of the cell cycle by inhibition of TopoII⁴ activity. Because this checkpoint seems to be absent in yeast, its existence was first described in mammals by demonstrating checkpoint bypass induced by caffeine or phosphatase inhibitors after treatment with TopoII catalytic inhibitors (5). Catalytic inhibitors, such as ICRF154, ICRF187, and ICRF193, inhibit ATPase activity of TopoII and stabilize the enzyme in the form of a closed clamp instead of stabilizing the TopoII/DNA-cleavable complex so that no DNA strand breaks are induced (6, 7). The decatenation checkpoint is distinct from the G₂ DNA damage checkpoint because it appears to be ATM independent and is not sustained by down-regulation of cyclin-dependent kinase 1/cyclin B1 activity (5, 8). Instead, it was demonstrated that decatenation checkpoint activation relies on ATR activity and nuclear exclusion of cyclin B1 (8). Because the decatenation checkpoint is not properly activated in BRCA1-mutant cells (8), it seems that a functional BRCA1 protein is also required in this checkpoint response. However, it is still unknown whether physical structures within chromosomes, such as chromosome tangles, are monitored by the TopoII-dependent decatenation checkpoint and whether other proteins help TopoII in this function. The precise target(s) of the ATR kinase is also unknown. Override of the decatenation checkpoint and progression into mitosis in the absence of TopoII activity could result in catenated chromatids before prophase, failed segregation of sister chromatids in metaphase-anaphase transition, and, finally, a high degree of genomic instability (9, 10, 11).

DNA Topos and helicases work together in many aspects of DNA metabolism where the individual strands of double-stranded DNA must be separated (12).

The Sgs1 protein, a yeast RecQ helicase, physically interacts with both TopoII and TopoIII (13, 14, 15), and their association would be important for facilitating decatenation of late-stage replicons (13, 16). Also, two of the human RecQ helicases, the Bloom syndrome protein (BLM) and RECQ5, physically interact with TopoIIIα, contributing to maintenance of genomic stability (17, 18). Mutations in some of the genes encoding RecQ helicases are implicated in human disorders such as WS and Bloom syndrome, caused by mutations in WRN and BLM, respectively (19), which are both characterized by genomic instability and cancer predisposition. Biological and biochemical evidence suggests that WRN helicase functions in multiple DNA metabolisms, including replication and recombination (19).

In this study, we analyzed the activation of the decatenation checkpoint and its contribution to the maintenance of genomic stability in WS cells.

Normal (SNW646) and WS (KO375 and DJG) LCLs were obtained from the International Registry of WS (Dr. George Martin, University of Washington, Seattle, WA). Normal LCL GM3572 and the WS LCL AG14426 as well as the normal primary (GM1215) and the WS fibroblasts (AG12795) were obtained from Coriell (Coriell Cell Repositories, Camden, NJ). The culture conditions of LCLs and fibroblasts have been described previously (20). The AG14426 and KO375 cell lines carry an Arg369>stop mutation that gives rise to a truncated protein. DJG is the compound heterozygote of Arg369>stop and deletion of nucleotides 2320–3056 (21). The WS1WRN was generated by transfection by electroporation of linearized pcDNA3.1WRN plasmid expressing the wt WRN cDNA. Cell lines conditionally expressing ATRkd have been described previously (22). Expression of ATRkd was achieved by the addition of 1 μg/ml doxycycline for 72 h. Considering the poor growth of the primary WS cells, it was necessary to largely use lymphoblastoid cell lines for our experiments.

Cells (1 × 10⁷) were lysed as described previously (23), and 40 μg of total proteins were subjected to SDS-PAGE. Immunoblots were performed with rabbit polyclonal antibodies against WRN (Novus Biochemicals; 1:4000), BRCA1 (Santa Cruz Biotechnology), or TopoII (Oncogene Research) followed by chemiluminescence detection by enhanced chemiluminescence plus (Amersham). The anti-TopoII antibody used in this study allows detection of both α and β forms. Equal loading and transfer were monitored by Ponceau red staining of the membrane or by reprobing the blots with anti-actin antibody.

In immunoprecipitation experiments, cells were lysed in a mild lysis buffer as described previously (23), and 0.5–2 mg of total proteins were immunoprecipitated using anti-BRCA1 (Oncogene Research) or anti-WRN (BD PharMingen) monoclonal antibodies. For λ-phosphatase treatment, BRCA1 immunoprecipitates were resuspended in a phosphatase buffer and incubated for 30 min with 300 units of λ-phosphatase (New England BioLabs) at 30°C and then boiled in 2× electrophoresis sample buffer before Western blotting analysis.

In all cases, MI was determined by counting 1000 cells/culture, as described previously (24). Localization of cyclin B1 by immunofluorescence was carried out as described previously (8) on mock-treated cells or cells exposed to 150 μm ICRF187 for 3 h.

Subpopulations of cells in S phase were labeled with 30 μg/ml BrdUrd (Sigma-Aldrich) for 2 h, and then 150 μm ICRF187 (Cardioxane; Chiron) was added for 4 h to induce the checkpoint response in labeled cells. Duplicate cultures from wt lymphoblasts were treated with 2 mm caffeine (Sigma-Aldrich) together with ICRF187 to induce a checkpoint override. The chromosomal instability derived from the decatenation checkpoint override was analyzed in BrdUrd-labeled cells harvested at their second mitosis after 21 and 25 h of recovery.

For the determination of the effects of a continuous inhibition of TopoII, normal and WS cells were exposed to 150 μm ICRF187 for 12, 18, and 24 h. For the evaluation of checkpoint override in wt cells, cultures were exposed for 8 h to 150 μm ICRF187 plus 2 mm caffeine and then washed and placed into ICRF187-containing medium. Cells were then harvested, fixed, and stained with bis-benzimide H33342 (Sigma) as described previously (20). A minimum of 1000 cells were scored for each experimental point, and statistical analysis of a minimum of three independent experiments was done by χ² test.

DNA damage was evaluated by comet assay (single cell gel electrophoresis) under denaturing conditions as described by Olive et al. (25). The analysis of induced damage was performed at 0, 6, 8, and 12 h; the addition of 30 μg/ml BrdUrd during the last 2 h before harvesting allowed us to determine whether cells with comets belong to the S phase. Immunodetection of BrdUrd incorporation was made according to our previously described procedure (24). A minimum of 200 cells were analyzed for each experimental point.

Cells treated with or without ICRF187 were harvested after 0, 6, 10, or 14 h; spread onto poly-l-lysine-coated slides; and fixed and immunostained with rabbit polyclonal anti-RAD51 (Oncogene Research) as described previously (20). For each time point, at least 200 nuclei were examined, and RAD51 foci were scored at ×100 magnification. Only nuclei showing >10 foci were considered positive.

To assess the involvement of WRN in the G₂ decatenation checkpoint, activation of the decatenation checkpoint was determined by measuring the M-phase progression in LCLs and primary fibroblasts from normal or WS individuals. In addition, WS LCLs complemented with wt WRN were used for the study. Functional complementation was verified by analyzing sensitivity to the drug camptothecin in a standard apoptotic assay as well as by Western blot (see Supplementary Fig. 1).² To induce the decatenation checkpoint we used ICRF187. After a 3-h treatment with different doses of ICRF187, wt cells already showed a significant reduction of the MI at the lower dose with a maximum of inhibition at 50 μm and saturation at the higher dose. Analysis of the kinetics of decatenation checkpoint activation, investigated after exposure to 150 μm ICRF187, showed that inhibition of the progression into mitosis was maximal at 3 h (Fig. 1, A and B), in agreement with the previously reported data (8). In contrast, WS cells did not display any mitotic inhibition at any dose of ICRF187 used and at any of the harvesting times analyzed (Fig. 1, A and B). This defect was completely restored upon introduction of a wt WRN cDNA (Fig. 1, A and B).

To verify the effect of the decatenation checkpoint override, wt cells were treated with 2 mm caffeine plus 150 μm ICRF187. As shown in Fig. 1,C, caffeine removed ICRF187-induced G₂ arrest in wt cells but did not affect progression into mitosis of WS cells, which had already escaped the decatenation checkpoint. In wt cells, caffeine treatment induced uncondensed and entangled mitotic figures [Fig. 1,D, b; a sign of premature entry into mitosis after failure of decatenation checkpoint activation (5)], which are similar to those observed in WS cells treated with ICRF187 (Fig. 1 D, d). Chromosome structures of wt cells treated with caffeine only were indistinguishable from those in cells treated with only ICRF187; chromosome structures of WS cells treated with ICRF187 were indistinguishable from those present in cultures treated with ICRF187 plus caffeine (data not shown).

Decatenation checkpoint activation has been shown to rely on nuclear exclusion of cyclin B1 (8). We therefore examined localization of cyclin B1 in wt and WS cells after triggering the decatenation checkpoint by ICRF187 treatment. We found that cyclin B1 was excluded from the nucleus in wt and complemented WS cells, but not in WS cells (Fig. 1 E). Also inhibition of Polo-like kinase (PLK) activity, a phenomenon recently associated with activation of the decatenation checkpoint (26), was defective in WS cells (data not shown).

The defect in the decatenation checkpoint activation observed in WS cells was not attributable to differences in TopoIl levels or activity as assessed by Western immunoblot analysis (Fig. 1 F) or TopoII assay (data not shown) and was consistent with our previously reported data (27).

These results provide direct evidence that the decatenation checkpoint was not properly activated in WS cells and that a functional WRN is required for the checkpoint activity.

ATR kinase and the BRCA1 protein have been shown to be essential for correctly establishing the decatenation checkpoint (8). It has been reported that ATR is able to phosphorylate BRCA1 after several genotoxic stresses (28, 29), but it is not known whether BRCA1 is also phosphorylated in response to decatenation checkpoint activation. Thus, we investigated the ability of ICRF187 to induce the phosphorylation of BRCA1 in wt, WS, and ATRkd cells.

Treatment with ICRF187 induced a bandshift in BRCA1 in wt cells (Fig. 2,A). This shift was absent in WS cells and in cells overexpressing ATRkd but was present in WS-complemented cells (Fig. 2,A). As expected, treatment with 30 J/m² UVC caused a BRCA1 bandshift in wt and WS cells, but not in ATRkd cells (Fig. 2,A). Moreover, phosphatase treatment of BRCA1 immunoprecipitates from mock-treated or ICRF187-exposed cells reversed the bandshift in wt cells, but not in WS or ATRkd cells, indicating that the observed mobility shift was attributable to phosphorylation (Fig. 2 B).

WRN, like BRCA1, is phosphorylated after replication arrest or several DNA-damaging treatments (23, 30), so we investigated whether WRN was phosphorylated as a consequence of the decatenation checkpoint activation. Treatment with ICRF187 failed to produce a detectable mobility shift of WRN in wt cells, whereas a slower-migrating form was readily detected after UVC treatment, suggesting that WRN is not phosphorylated in response to catenated DNA (Fig. 2 C). It remains to be determined whether WRN phosphorylation is indeed unaltered.

Because WRN seems to be involved in decatenation checkpoint activation, and given that Topos may interact with RecQ helicases (12), we sought to determine whether WRN and TopoII coimmunoprecipitate in wt cells after decatenation checkpoint activation. As shown in Fig. 3 D, TopoII was detected in WRN immunoprecipitates after ICRF187 treatment. Because the available TopoII antibody does not work well in either immunoprecipitation or immunofluorescence, we could not test the presence of WRN in TopoII immunoprecipitates or a possible WRN/TopoII colocalization after decatenation checkpoint induction.

These data indicate that ATR-dependent phosphorylation of BRCA1 is associated with decatenation checkpoint activation and that a functional WRN is required for decatenation checkpoint-dependent phosphorylation of BRCA1.

We then investigated whether failure of the decatenation checkpoint could correlate with enhanced chromosomal instability in WS cells. To test this hypothesis, metaphase preparations from ICRF187-treated wt and WS cells, as well as ICRF187-treated wt cells in which the checkpoint was reversed by caffeine, were analyzed for chromosomal aberrations. Examination of the chromosomal damage caused by an override of the decatenation checkpoint in first mitosis cells (i.e., cells harvested immediately after treatment) is made difficult due to improper chromatid condensation and decatenation. To overcome this problem, we analyzed chromosomal aberrations in cells in second mitosis. A subpopulation of S-phase cells was pulse-labeled with BrdUrd and ICRF187, and chromosomal damage was scored only in the labeled population. In WS cells, checkpoint override did cause chromosomal instability, but the number of chromosomal aberrations was only about 2-fold the control levels (Table 1). Interestingly, caffeine-mediated checkpoint override in wt cells resulted in less chromosomal damage with respect to that observed in WS cells. Because increased chromosomal damage after checkpoint override could be masked by apoptotic cell death, we analyzed apoptosis from parallel cultures. Apoptotic cell death was not triggered by pulse exposure to ICRF187 in either WS cells or wt cultures where checkpoint was inactivated by caffeine treatment (Table 1).

We performed the same analysis in cells induced to overexpress ATRkd. Induced cells also showed an elevated yield of chromosomal damage in the absence of any treatment (Supplementary Table 1),² in agreement with the findings of Deming et al. (8). Treatment with ICRF187 resulted in a ∼4-fold increment of the chromosomal damage over the control and also resulted in a higher rate of apoptotic cell death (Supplementary Table 1).²

These results suggest that the mere deficiency of the decatenation checkpoint function does not result in a significant induction of chromosomal instability unless other surveillance systems (i.e., WRN or ATR) are leaky or absent.

Override from the decatenation checkpoint did not result, per se, in large chromosomal instability unless other major surveillance systems are defective (i.e., ATR) Thus, we wondered whether sustained TopoII inhibition could result in enhanced apoptotic cell death in cells that escaped the decatenation checkpoint. Because catalytic inhibition of TopoII has been reported not to affect the M-G₁ or the G₁-S transition (10), we focused our analysis on the effect of the progression through S phase. We treated cultures with 50 or 150 μm ICRF187 and examined them 12, 18, or 24 h later (Fig. 3, A and B, respectively). We found that continuous TopoII inhibition induced apoptotic cell death in WS cells (Fig. 3, A and B). This effect was time dependent; in fact, apoptosis appeared starting at 18 h and reached maximum value at 24 h. The level of apoptotic cells was at about 15% when we treated cells with 50 μm ICRF187 but became about 35% at 150 μm ICRF187, showing that this phenomenon was dose dependent. It is also noteworthy that transfection with wt WRN cDNA restored resistance to ICRF187-induced apoptosis. Because wt cells efficiently trigger the checkpoint in response to ICRF187 treatment, we forced override of the decatenation checkpoint by a pulse treatment with caffeine. After 8 h of caffeine treatment, wt cells were placed into a medium containing only ICRF187 to avoid permanent exposure to caffeine, which could result in the inhibition of other surveillance pathways. We found that wt cells that escaped the decatenation checkpoint by caffeine and were treated continuously with ICRF187 underwent apoptotic cell death (∼7%, Fig. 3,C), but to a lesser extent than that seen in WS cells, also taking into account that under our experimental conditions, we induced release from the checkpoint only in ∼50% of the total wt cellular population (Fig. 3 D).

These results suggest that the elevated rate of apoptosis observed in WS cells after TopoII inhibition might correlate with the role that WRN carries out in the cell, possibly in the subsequent S phase.

Because we found that sustained inhibition of TopoII activity in the next round of replication after decatenation checkpoint failure triggered apoptotic cell death, we analyzed the degree of DNA damage induced in S phase by ICRF187 treatment in cells that escape from the decatenation checkpoint. To visualize damaged cells belonging to the S phase, we exposed cells to BrdUrd. DNA damage was analyzed at 0, 6, 8, and 12 h after 150 μm ICRF187 treatment by comet assay (Fig. 4,A). We found that continuous inhibition of TopoII produced DNA damage in a time-dependent manner in WS cells, but not in wt cells. On the other hand, when we analyzed comets induced by continuous ICRF187 exposure followed by an 8-h pulse treatment with caffeine, we detected a time course of induced DNA damage in wt cells that was similar to that observed in WS cells, although at a reduced level. This is consistent with the fact that only a subpopulation of wt cells evaded the decatenation checkpoint because of the pulse exposure to caffeine (Fig. 4,A). Interestingly, about 80% of cells with comets belonged to the S phase as showed by BrdUrd incorporation in either WS or caffeine-treated wt cells (Fig. 4,B). Consistent results were obtained analyzing by the subnuclear relocalization of the RAD51 recombinase, which is a marker of an ongoing double-strand break repair in the S phase. RAD51 relocalization was observed only in ICRF187-treated WS cells or in wt cells in which the decatenation checkpoints were overridden by caffeine, but not in wt cells exposed only to ICRF187 or in mock-treated controls (Fig. 4, C and D). Interestingly, the percentage of RAD51-positive nuclei roughly matched that of the cells showing BrdUrd-positive comets as shown in Fig. 4 A.

These findings suggest that DNA damage, notably double-strand breaks, are formed as a consequence of decatenation checkpoint failure when catenated DNA attempts to be replicated in the absence of an active TopoII.

In addition, to monitor the completion of a certain cellular function and to sense DNA damage and replication arrest, a specific checkpoint exists that supervises correct catenation status of chromatids before mitotic entry (5). Activation of this decatenation checkpoint involves the function of ATR and BRCA1 and nuclear exclusion of cyclin B1 (8). However, the manner in which the catenated status of chromatids is sensed, as well as the downstream target(s) of ATR and the role for BRCA1, is currently unknown. Here, we demonstrate that a functional WRN protein is required for activation of the G₂ decatenation checkpoint.

The helicase activity of WRN together with the activity of TopoII could serve as a sensor to monitor catenation status of chromatids, leading to the activation of the checkpoint cascade. Consistent with this hypothesis, we found that WRN and TopoII coimmunoprecipitated after decatenation checkpoint activation and that phosphorylation of BRCA1, one of the elements of decatenation checkpoint function, is absent in WS cells. In yeast, the RecQ helicase Sgs1 has been proposed to collaborate with TopoII in the decatenation of late-stage replicons at sites of converging forks (13), and bacterial RecQ together with TopoIII may present an active catenase/decatenase activity (13, 31). It is possible that WRN and TopoII could represent in human cells a functional analogue of Sgs1 and TopoII in yeast. Accordingly, it might be possible that the decatenation checkpoint function senses decatenation status of very late-stage replicons, arresting the cell cycle if these replicons are still catenated, hence triggering the checkpoint response. From this point of view, it is not surprising that the upstream kinase involved in the decatenation checkpoint activity is ATR and not ATM because ATR is the key kinase in response to stress at the replication fork level (32).

Despite the risk of cell cycle progression in the presence of tangled and incompletely decatenated chromatids, little is known about the direct contribution of the decatenation checkpoint to genetic stability. We provided evidence that failure of the decatenation checkpoint is not sufficient per se to greatly increase genomic instability, at least at the chromosomal level, because chromosomal damage is not overtly enhanced after ICRF187 treatment in either WS cells or wt cells that artificially escaped the checkpoint. On the contrary, BRCA1 mutations or expression of a dominant-negative form of ATR, two other conditions associated with defects in the decatenation checkpoint, results in a phenotype suggestive of inappropriate mitotic entry, with several chromosomal abnormalities and features of mitotic catastrophe (8). Our results suggest that this widespread genomic instability probably does not derive directly from entering into mitosis with insufficiently decatenated chromatids, but rather from the combined effects of failure of multiple surveillance pathways because ATR and BRCA1 are both involved in several cell cycle arrest pathways (32, 33). On the contrary, WS cells apparently do not have other major checkpoint defects (20, 34), thus allowing a more accurate analysis of the contribution of decatenation checkpoint failure to gross chromosomal instability.

Consistently, ICRF187 treatment of ATRkd cells led to a greater enhancement of chromosomal damage over the mock-treated control (Supplementary Table 1²) than that observed in WS cells.

When sustained TopoII inhibition was combined with the decatenation checkpoint failure, we found that DNA strand breaks were formed in replicating cells, leading to the activation of DNA repair and ultimately to apoptotic cell death, which was markedly elevated in the absence of an active WRN protein. Because such a large apoptotic response was not found after pulse treatment with ICRF187⁵ (Table 1) and because of a selective induction of DNA strand breakage in cells undergoing DNA replication with insufficiently decatenated chromatids, it is possible that TopoII and WRN activities are required to deal with structural problems before or during DNA replication. In particular, TopoII could decatenate still-catenated chromatids of cells that have overridden the decatenation checkpoint after or during DNA replication, avoiding DNA breakage. Indeed, a function of the decatenase activity of TopoII throughout the S phase has been described previously (31), and a delay of the S phase after catalytic inhibition of TopoII has been also been reported (10). On the other hand, DNA breakage resulting from mitotic progression in the presence of tangled chromatids could be handled by the DNA repair systems before S phase. In the absence of an active TopoII, extensive DNA breakage is created in replicating cells, requiring massive replication-associate DNA repair. Such a pathway can be carried out by recombination and could very well rely on the presence of an active WRN protein (20, 35). In the concurrent absence of TopoII and WRN activities, unrepaired or misrepaired DNA breakage could cause apoptotic cell death. Supporting this possibility, WRN deficiency determines a phenotype of enhanced apoptosis as a consequence of the induction of DNA breakage selectively in replicative cells (20).

Taken together, our results integrate WRN and TopoII in the process of sensing incomplete catenated chromatids to trigger the checkpoint response and the consequent BRCA1 phosphorylation (Fig. 4 E) and further implicate WRN in the correct handling of DNA strand breakage resulting from replicating incomplete catenated chromatids. Our findings also suggest that failure of the decatenation checkpoint could contribute to genomic instability through association with mutations in other “caretaker” genes, such as ATR, BRCA1 or WRN, rather than by itself.

We are grateful to Drs. S. Handeli and S. L. Schreiber for providing the cells expressing ATRkd.