Rapid PIKK-Dependent Release of Chk1 from Chromatin Promotes the DNA-Damage Checkpoint Response

Current Biology 16, 150–159, January 24, 2006 ª2006 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2005.11.066

Article Rapid PIKK-Dependent Release of Chk1 from Chromatin Promotes the DNA-Damage Checkpoint Response Veronique A.J. Smits,1,2,3,* Philip M. Reaper,1,3,4 and Stephen P. Jackson1,* 1 The Wellcome Trust and Cancer Research UK Gurdon Institute Tennis Court Road Cambridge CB2 1QN United Kingdom 2 Department of Cell Biology and Genetics Erasmus MC P.O. Box 1738 3000 DR Rotterdam The Netherlands

Summary Background: Checkpoint signaling pathways are of crucial importance for the maintenance of genomic integrity. Within these pathways, the effector kinase Chk1 plays a central role in mediating cell-cycle arrest in response to DNA damage, and it does so by phosphorylating key cell-cycle regulators. Results: By investigating the subcellular distribution of Chk1 by cell fractionation, we observed that around 20% of it localizes to chromatin during all phases of the cell cycle. Furthermore, we found that in response to DNA damage, Chk1 rapidly dissociates from the chromatin. Significantly, we observed a tight correlation between DNA-damage-induced Chk1 phosphorylation and chromatin dissociation, suggesting that phosphorylated Chk1 does not stably associate with chromatin. Consistent with these events being triggered by active checkpoint signaling, inhibition of the DNA-damage-activated kinases ATR and ATM, or siRNA-mediated downregulation of the DNA-damage mediator proteins Claspin and TopBP1, impaired DNA-damage-induced dissociation of Chk1 from chromatin. Finally, we established that Chk1 phosphorylation occurs at localized sites of DNA damage and that constitutive immobilization of Chk1 on chromatin results in a defective DNA-damage-induced checkpoint arrest. Conclusions: Chromatin association and dissociation appears to be important for proper Chk1 regulation. We propose that in response to DNA damage, PIKKdependent checkpoint signaling leads to phosphorylation of chromatin-bound Chk1, resulting in its rapid release from chromatin and facilitating the transmission of DNA-damage signals to downstream targets, thereby promoting efficient cell-cycle arrest.

*Correspondence: [email protected] (V.A.J.S.); s.jackson@ gurdon.cam.ac.uk (S.P.J.) 3 These authors contributed equally to this work. 4 Present address: Vertex Pharmaceuticals, 88 Milton Park, Abingdon, Oxfordshire, OX14 4RY, United Kingdom.

Introduction DNA-damage checkpoints are triggered by DNA lesions or by inhibition of DNA synthesis, and they lead to the elaboration of various cellular events such as cell-cycle arrest, DNA repair, and apoptosis. ATR and ATM, members of the phosphoinositide 3-kinase-related protein kinase (PIKK) family, are key components of the DNAdamage checkpoint response and phosphorylate and activate the effector kinases Chk1 and Chk2 [1]. These effector kinases then regulate DNA-damage-induced arrest at specific stages of the cell cycle by targeting key cell-cycle regulators. Although the ATM-Chk2 pathway is primarily activated by ionizing radiation (IR) [2], whereas ultraviolet (UV) light and stalled replication forks trigger the ATR-Chk1 pathway [3], recent studies have shown that Chk1 is also activated in response to IR in an ATM-dependent manner [4–6]. In addition to the PIKKs, which directly regulate Chk1 by phosphorylation, several other checkpoint proteins are required for efficient Chk1 activation. The early events after induction of DNA damage include the independent recruitment of ATR and an RFC-related protein, Rad17, to RPA-coated single-stranded DNA. Subsequently, the Rad9-Rad1-Hus1 (9-1-1) complex is recruited to chromatin by the Rad17-RFC complex, and ATR phosphorylates Rad17 in a Hus1-dependent manner [7, 8]. Together, these events are thought to regulate Chk1 phosphorylation, because Chk1 phosphorylation is largely abrogated in cells deleted for Hus1 or Rad17 [9, 10]. Recent work has also implicated the DNA-damage mediator proteins BRCA1 [11], TopBP1 [12], MDC1/NFBD1 [13], and Claspin [14, 15] in controlling Chk1 regulation. Although it is not entirely clear how these mediators function, it has been hypothesized that they might recruit Chk1 to ATM/ATR and thus provide a platform for Chk1 phosphorylation to take place. Several studies in human cells and Xenopus egg extracts have focused on how Claspin regulates Chk1. For example, Claspin was shown to bind Chk1, and this interaction was strongly stimulated by genotoxic stress, and mutant Claspin that cannot bind Chk1 was unable to mediate DNA-damage-induced activation of Chk1, indicating that Claspin binding is required for Chk1 activation [16, 17]. The C terminus of Chk1 contains several conserved SQ motifs that conform to consensus sites for phosphorylation by the ATM and ATR kinases. Two of these sites, serine 317 and serine 345 in human Chk1, have been shown to be phosphorylated after genotoxic stress, and these have been found to be phosphorylated by ATR in vitro and phosphorylated in an ATR-dependent manner in vivo [3, 18]. DNA-damage-induced phosphorylation has been suggested to regulate Chk1 function, because phosphorylated Chk1 has higher kinase activity than the unphosphorylated protein, and Chk1 mutants in which serines 317 and 345 were replaced by alanines showed poor activation in response to

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Figure 1. DNA-Damage-Induced Dissociation of Chk1 from Chromatin (A) U2OS cells were left untreated or were treated with 50 J/m2 of UV. Two hours later, cells were fractionated as described in the Experimental Procedures. Cytoplasmic proteins (S1) and soluble nuclear proteins (S2) were pooled to one soluble fraction; P2 is the chromatin fraction. From each fraction, we loaded equal amounts of proteins onto SDS-PAGE and analyzed them by immunoblotting for Chk1. Two different exposures of the Chk1 immunoblot are shown. Subsequently, blots were stripped and reprobed for the DNA replication-licensing chromatin protein Orc2, which was used as a loading control for chromatin samples. Numbers at the bottom of the sample lanes represent the relative Chk1 protein level compared to that of the untreated control (after normalization of the levels of the loading control Orc2). (B) U2OS cells were treated and fractionated as in (A). Chk1 was detected in the ‘‘cytoplasmic’’ fraction (S1), but because other DNA-damage-checkpoint factors that we detected in this fraction are thought to primarily localize to the nucleus (Figure 2C and data not shown) [28], we think this predominantly reflects rapid leakage of certain proteins from the nucleus during fractionation. (C) U2OS cells were synchronized at the G1/S transition with a double thymidine block, after which they were released. At the indicated times, cells were harvested and fractionated as above. As a control, asynchronous U2OS cells were left untreated or were treated with 50 J/m2 of UV, and they were then fractionated 2 hr after treatment. Fractions S1 and S2 were pooled. Arrows point out Chk1, and the asterisk indicates a cross-reacting factor with a slightly higher apparent molecular weight. The cell-cycle stage of the synchronized cells was determined by propidium iodide (PI) staining and FACS analysis. (D) U2OS cells were treated with 50 J/m2 of UV and then incubated for the indicated times before being fractionated and analyzed as above. (E) U2OS cells were treated with the indicated doses of UV, and 2 hr later, were fractionated and analyzed as above.

DNA damage [18]. Significantly, there are data indicating that the Chk1 C terminus plays an inhibitory role by binding the kinase domain of the same molecule, and DNAdamage-induced phosphorylation of the C terminus has been suggested to release this autoinhibition [19]. Phosphorylation of Chk1 could also facilitate its association with other proteins, thereby contributing to the checkpoint response. For example, it has been shown that fission yeast and human Chk1 can bind 14-3-3 proteins and that this association is stimulated in response to DNA damage [20, 21]. Such interactions could also potentially regulate the cellular localization of Chk1, as suggested by Jiang et al. [21]. Here, we describe an additional mechanism that contributes to the regulation of Chk1: Chk1 binds to chromatin in unperturbed cells but dissociates from chromatin in response to DNA damage. Furthermore, we establish that Chk1 chromatin dissociation depends on checkpoint signaling and appears to be regulated by Chk1 phosphorylation, which takes place on chromatin in close proximity to sites of DNA damage. Finally, we show that immobilization of Chk1 on chromatin diminishes the

efficiency of the G2 checkpoint arrest, suggesting that Chk1 chromatin dissociation facilitates transmission of the DNA-damage signal to downstream targets. Results Chk1 Associates with Chromatin and Is Released in Response to DNA Damage To investigate the cellular localization of Chk1, we fractionated extracts of asynchronously growing U2OS cells by a centrifugation-based method that was developed by Me´ndez and Stillman [22] and has been used to study the localization of the DNA-damage signaling proteins Rad17 and Rad9 [8]. Thus, we obtained fractions of cytoplasmic proteins (S1), soluble nuclear proteins (S2), and a chromatin-enriched fraction (P2). Chk1 was detected in the soluble fractions and in the insoluble P2 fraction (Figure 1A), and treatment of the nuclei with micrococcal nuclease [22] resulted in a considerable decrease of Chk1 in the insoluble fraction, which indicates that Chk1 is indeed associated with chromatin (Figure S1 in the Supplemental Data available with this article

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online). We estimate around 20% of the total cellular Chk1 pool to be chromatin-associated (Figure 1A). Strikingly, in response to UV treatment, the amount of chromatin-bound Chk1 was substantially reduced, suggesting that UV treatment triggers dissociation of Chk1 from chromatin (equivalent results were obtained with several Chk1 antibodies; data not shown). Furthermore, the UVinduced slower migrating forms of Chk1 were clearly observed in the soluble fraction but not in the chromatin fraction (Figure 1A). Indeed, when we immunoblotted with phosphospecific antibodies that detect DNA-damage-induced PIKK-mediated phosphorylation of Chk1 on Ser345 or Ser317, the phosphorylated forms of Chk1 only appeared in the soluble fractions (Figure 1B), confirming the essential absence of phosphorylated Chk1 on chromatin. Importantly, the Chk1 chromatin association and effect of UV treatment were also observed in HeLa cells (data not shown) and in mouse embryonic fibroblasts (MEFs; see below). Thus, the dissociation of Chk1 from chromatin in response to DNA damage is not cell-type-specific and is conserved from mouse to man. Because there is evidence for Chk1 functioning during the unperturbed cell cycle [5, 23], it seemed possible that Chk1, like ATR [24], is primarily recruited to chromatin during S phase. The observed reduced association of Chk1 with chromatin in response to UV damage could, therefore, simply be an indirect consequence of the DNA-damage-induced inhibition of cell-cycle progression. To investigate the potential cell-cycle phase dependency of Chk1 association with chromatin, we synchronized U2OS cells at the G1/S transition by a double thymidine block, released them from this block, and, at various time points after the release, harvested them for chromatin fractionation. Although thymidine treatment— which is known to induce DNA damage—resulted in slight phosphorylation of Chk1 in the soluble fraction, the levels of Chk1 on chromatin did not change markedly during progression through the cell cycle (Figure 1C). Blocking U2OS cells at the metaphase/anaphase transition by nocodazole treatment, followed by their release into the cell cycle and analysis of Chk1 chromatin association at different time points, gave equivalent results (Figure S2). These findings indicate that in these cells, Chk1 associates with chromatin throughout various phases of the cell cycle, and they show that its DNAdamage-induced dissociation is unlikely to reflect cellcycle arrest (also, see below). We next investigated the timing of the DNA-damageinduced release of Chk1 from chromatin by treating U2OS cells with UV light and fractionating cell extracts at various time points after treatment. This revealed that Chk1 is very rapidly released from chromatin in response to UV (Figure 1D). Indeed, even when cells were harvested immediately after UV treatment (t = 5 min), Chk1 was already phosphorylated at Ser345 to a significant degree and, moreover, had clearly already begun dissociating from chromatin. These results indicate that there is a close correlation between Chk1 phosphorylation and its chromatin dissociation. Treating U2OS cells with different doses of UV supported this conclusion (Figure 1E). Thus, Chk1 was phosphorylated even at very low UV doses (2 J/m2), and at such low doses, we also observed significantly reduced levels of Chk1 in the chromatin fraction (Figure 1E). In addition to revealing that Chk1

chromatin dissociation is efficiently and rapidly triggered by UV treatment, these results provide further support for the conclusion that Chk1 chromatin dissociation is not an indirect consequence of downstream checkpoint events, such as altered cell-cycle distribution. Consistent with Chk1 chromatin release being a general response to genotoxic stress, we found that, similar to UV, treating U2OS cells with Hydroxyurea (HU) also resulted in an induction of phosphorylated Chk1 in the soluble fraction and Chk1 chromatin dissociation (Figure 2A). Furthermore, soluble Chk1 also underwent dose-dependent phosphorylation after IR treatment, albeit to a lesser extent than after UV treatment, and the amount of chromatin-bound Chk1 decreased accordingly after IR, again revealing a close correlation between Chk1 phosphorylation and its chromatin association status (Figures 2A and 2B). Dissociation of Chk1 from Chromatin Depends on Active DNA-Damage Checkpoint Signaling The above treatments led to activation of signaling pathways controlled by the PIKKs ATM and ATR. To ascertain whether Chk1 chromatin release after DNA damage depends on the activity of these PIKKs, we incubated U2OS cells with the PIKK inhibitors wortmannin and caffeine before treating them with UV light. Although wortmannin and caffeine did not affect the association of Chk1 with chromatin in undamaged cells, they completely prevented the normal UV-induced dissociation of Chk1 from chromatin and Chk1 phosphorylation in the soluble fractions (Figure 2C). Furthermore, downregulation of ATR protein levels by RNA interference (RNAi) also diminished the dissociation of Chk1 from chromatin in response to UV (data not shown). In addition, IR-induced phosphorylation and chromatin dissociation of Chk1 were largely prevented when cells were preincubated with the specific ATM inhibitor KU-55933 [25] (Figure 2D). Together, these results indicate that, although the association of Chk1 with chromatin in untreated cells does not require ATM or ATR activity, these PIKKs are needed for the DNA-damage-induced dissociation of Chk1 from chromatin. Experiments with fibroblasts from Hus1 null mice have shown that ATR-dependent phosphorylation of Chk1 depends on the 9-1-1 complex [9]. Because our data suggested that the affinity of Chk1 for chromatin is regulated by its DNA-damage-induced phosphorylation, we examined UV-induced chromatin dissociation of Chk1 in Hus12/2p212/2 cells. Although UV treatment resulted in Chk1 phosphorylation in control Hus1+/+p212/2 MEFs, this phosphorylation of Chk1 was not seen in Hus12/2p212/2 MEFs (Figure 3A). Moreover, whereas Chk1 chromatin association decreased after UV treatment of the control cells, this decrease was much less apparent in Hus12/2p212/2 MEFs. These data indicate that, as in human cells, Chk1 dissociation after DNA damage in mouse cells parallels the phosphorylation status of the protein. Recent studies have shown that the DNA-damage mediator proteins TopBP1 and Claspin control DNAdamage-induced checkpoint events. Thus, TopBP1 controls Chk1 phosphorylation and is required for an optimal G2/M arrest in response to DNA damage [12], whereas Claspin regulates Chk1 activation in both

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Figure 2. PIKK Activity Is Required for Dissociation of Chk1 from Chromatin (A) U2OS cells were treated with 50 J/m2 of UV (2 hr), 20 Gy of IR (2 hr), or 1 mM of HU (24 hr), after which they were fractionated as described. Fractions S1 and S2 were pooled. We loaded equal amounts of protein and analyzed them by immunoblotting with the indicated antibodies. Actin and Orc2 were used as loading controls for the soluble and chromatin fractions, respectively. (B) U2OS cells were treated with the indicated doses of IR, and 1 hr later they were fractionated and analyzed as above. (C) U2OS cells were preincubated in the absence or presence of 100 mM wortmannin and 10 mM caffeine (Wort/Caff) for 15 min. Then the cells were left untreated or were treated with 80 J/m2 of UV, and they were fractionated after 2 hr of recovery. Grb2 and Orc2 were used as loading controls for cytoplasmic and chromatin samples, respectively. Arrows point out the protein of interest, and the asterisk indicates a cross-reacting protein. (D) U2OS cells were preincubated in the absence or presence of 10 mM KU-55933 (ATM inhibitor) for 30 min. Then the cells were untreated or treated with 25 Gy of IR, and after 1 hr of recovery, they were fractionated and analyzed as in (A). Numbers at the bottom of the sample lanes represent the relative Chk1 protein level compared to that of the untreated control (after the levels of the loading control Orc2 were normalized).

Xenopus and mammalian cells [14, 15]. When studying these proteins, we found that TopBP1 localized exclusively in the chromatin fraction (Figure 3B), whereas, in line with previous reports [21, 24], Claspin partitioned essentially evenly in the soluble and chromatin fractions

(Figure 3C). Notably, whereas siRNA-mediated downregulation of TopBP1 had essentially no effect on the Chk1 chromatin association in nonirradiated cells, it markedly decreased both DNA-damage-induced Chk1 phosphorylation and Chk1 chromatin dissociation (Figure 3B). Figure 3. Chk1 Chromatin Dissociation Depends on DNA-Damage Signaling (A) Hus1+/+p212/2 and Hus12/2p212/2 MEFs were untreated or treated with 50 J/m2 UV and fractionated 2 hr afterward. Fractions S1 and S2 were pooled. We loaded equal amounts of protein and analyzed them by immunoblotting with the indicated antibodies. (B) U2OS cells were oligofected with siRNA duplexes directed against Luciferase or TopBP1, and 72 hr later, the cells were treated with 50 J/m2 of UV or left untreated. After 2 hr recovery, cells were fractionated, and the S1 and S2 fractions were pooled. We loaded equal amounts of protein and analyzed them by immunoblotting for TopBP1, Chk1, and Orc2. (C) U2OS cells were oligofected with siRNA duplexes directed against Luciferase or Claspin. After 48 hr, the cells were treated, fractionated, and analyzed as described in (B). The arrow points to Chk1, and the asterisk indicates a cross-reacting protein.

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Similarly, downregulation of Claspin did not affect chromatin binding of Chk1 in undamaged cells but impaired Chk1 phosphorylation and chromatin dissociation after UV treatment (Figure 3C; the effects of Claspin downregulation were less dramatic than those caused by TopBP1 downregulation, but this is mainly due to the Claspin siRNA duplexes being less efficient in depleting the protein than those targeting TopBP1). In addition, IRand UV-induced phosphorylation and chromatin dissociation of Chk1 was less efficient in NBS1-LBI cells (data not shown), consistent with the described requirement for the Mre11/Rad50/Nbs1 complex in ATM signaling and a subset of ATR signaling events [6, 26, 27]. Taken together with other data, these results establish that DNA-damage-induced dissociation of Chk1 from chromatin depends specifically on checkpoint proteins that control Chk1 phosphorylation and activation, and they show that functional DNA-damage signaling is required for Chk1 chromatin dissociation. Link between Chk1 Phosphorylation and Its Chromatin Dissociation The above results raised the possibility that Chk1 phosphorylation directly triggers its chromatin dissociation. To explore this idea, we analyzed a Flag-tagged Chk1 mutant in which Ser317 and Ser345—the two known sites to undergo DNA-damage-induced PIKK-mediated phosphorylation [3, 18]—were mutated to alanine. We transfected the mutant (S317A/S345A) and wild-type (WT) Flag-Chk1 constructs into U2OS cells, treated these with UV, and subsequently fractionated the extracts. As shown in Figure 4A, although UV treatment resulted in the dissociation of Flag-Chk1 WT from chromatin, the extent of this dissociation was not as great as that observed for the endogenous Chk1 protein. Nevertheless, we reproducibly found that the degree of chromatin dissociation displayed by Flag-Chk1 WT was greater than that displayed by the S317A/S345A mutant. It is noteworthy that this Chk1 mutant still exhibited residual levels of chromatin dissociation (Figure 4A), and we suspect that this reflects the existence in human Chk1 of additional PIKK consensus (S/T-Q) sites that are phosphorylated in response to DNA damage in the wild-type or S317A/S345A mutant context (Figure S3 reveals that the S317A/S345A mutant still displayed residual phosphorylation after UV treatment; also see Discussion). By contrast, there was no reproducible difference in the degree of chromatin dissociation displayed by Flag-Chk1 WT and a derivative bearing an Ala substitution for Asp130 (D130A), which inactivates the kinase domain [18] (Figure 4B). Taken together, these results suggest that DNA-damage-induced dissociation of Chk1 from chromatin does not require Chk1 kinase activity but is at least in part regulated by PIKK-mediated phosphorylation of Chk1 on Ser317 and/or Ser345, with other PIKK-mediated Chk1 phosphorylation events potentially contributing to the efficiency of chromatin dissociation. Phosphorylation of Chk1 Occurs at Localized Sites of UV Damage DNA-damage-induced phosphorylation of many proteins by ATM and ATR is facilitated by their physical association with sites of DNA damage [8, 28–30]. We

Figure 4. Chk1 SQ Motif Phosphorylation and DNA-DamageInduced Chromatin Dissociation (A) U2OS cells were transfected with Flag-Chk1 WT or Flag-Chk1 S317A/S345A expression plasmids. After 24 hr, the cells were left untreated or were treated with UV (50 J/m2), and they were fractionated as described after 2 hr of recovery. We pooled the S1 and S2 fractions, loaded equal amounts of protein, and analyzed these by immunoblotting for Flag and Orc2. Numbers at the bottom of the sample lanes represent the relative Chk1 protein level compared to that of the untreated control (after levels of the loading control Orc2 were normalized). (B) U2OS cells were transfected with Flag-Chk1 WT or Flag-Chk1 D130A (kinase-inactive) expression plasmids and treated, fractionated, and analyzed as described in (A).

therefore considered the possibility that Chk1 is also phosphorylated at sites of damage and that the release of phosphorylated Chk1 from chromatin provides a mechanism to facilitate its interaction with its downstream substrates. Although the above data were consistent with such a model, they did not establish whether Chk1 phosphorylation and chromatin dissociation are sequential events. To address this issue, we generated a fusion of Flag-Chk1 with the chromatin component histone H2B [31], and this fusion resulted in an immobile protein that we found to be exclusively incorporated into the P2 chromatin fraction (Figure 5B). Importantly, when we treated U2OS cells expressing either Flag-Chk1 WT or H2B-Flag-Chk1 with UV light and prepared wholecell extracts (WCE) from these, we found that UV treatment resulted in increased phosphorylation of both wild-type and H2B-tagged Flag-Chk1 on the Ser317 and Ser345 Chk1 epitopes (Figure 5A and data not shown; phosphorylation of endogenous Chk1 was visible in cells expressing H2B-Flag-Chk1 but at lower levels than the fusion protein, presumably reflecting the higher amounts of H2B-Flag-Chk1 expressed). Moreover, whereas UV treatment resulted in phosphorylation of Flag-Chk1 and its dissociation from chromatin, phosphorylated H2B-Flag-Chk1 remained bound to the chromatin following UV treatment (Figure 5B). Thus, Chk1

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Figure 5. Chk1 is Phosphorylated at Sites of DNA Damage (A) U2OS cells were transfected with Flag-Chk1 WT (WT) or H2B-Flag-Chk1 (H2B) expression plasmids. After 48 hr, the cells were left untreated or were treated with UV (50 J/m2), and WCE were prepared 2 hr after the treatment. Equal amounts of protein were loaded and analyzed by immunoblotting for Flag or pSer317Chk1. (B) U2OS cells were transfected with Flag-Chk1 WT (WT) or H2B-Flag-Chk1 (H2B) expression plasmids. After 48 hr, the cells were left untreated or were treated with UV (50 J/m2). Two hours after treatment, cells were fractionated as described, and the S1 and S2 fractions were pooled. Equal amounts of protein were immunoblotted for the indicated epitopes. (C) U2OS cells were transfected with GFP-FlagChk1 (GFP) or H2B-GFP-Flag-Chk1 (H2B-GFP) expression plasmids and treated and analyzed as in (A). (D) U2OS cells were transfected with GFP-FlagChk1 (GFP) or H2B-GFP-Flag-Chk1 (H2B-GFP) expression plasmids and treated and analyzed as in (B). (E) U2OS cells were transfected with GFP-FlagChk1 or H2B-GFP-Flag-Chk1 expression plasmids. Forty-eight hours after transfection, cells were left untreated, or they were covered with a 5 mm filter and subjected to UV light (65 J/ m2) to induce local UV damage. After 1 hr, cells were fixed and immunostained with pSer317Chk1 and p62 antibodies. p62 staining identified the sites of local UV damage.

can indeed be phosphorylated when being bound to chromatin. To establish whether Chk1 is only phosphorylated in proximity to sites of DNA damage, we fused Flag-Chk1 and H2B-Flag-Chk1 to GFP to visualize these proteins by fluorescence microscopy. Importantly, both GFPFlag-Chk1 and H2B-GFP-Flag-Chk1 were phosphorylated in response to DNA damage (Figure 5C), and the phosphorylated form of H2B-Flag-Chk1 was detected solely in the chromatin fraction (Figure 5D). We treated cells expressing the above fusion proteins with UV light in the presence of an isopore filter—thereby generating UV damage within small areas of the nucleus—and subsequently analyzed the cells for GFP fluorescence and also immunostained them for Chk1-Ser317 phosphorylation and the p62 subunit of TFIIH, a complex that is known to accumulate at sites of UV damage [32]. Strikingly, whereas local UV damage induced phosphorylation of Chk1 throughout the nucleus in cells expressing

GFP-Flag-Chk1, phosphorylated Chk1 was essentially restricted to sites of local UV damage in cells expressing immobile H2B-GFP-Flag-Chk1, despite the fact that the protein itself was distributed uniformly throughout the nucleus as shown by GFP fluorescence (Figure 5E; in these cells, residual phosphorylated Chk1 can also be detected throughout the nucleus at higher laser power and likely represents endogenous, nonimmobilized Chk1). In addition, pre-extraction [33] of cells expressing GFP-Chk1 resulted in a total removal of phosphorylated Chk1, confirming the absence of phosphorylated Chk1 on chromatin. By contrast, after the pre-extraction procedure, spots of phospho-Chk1 were still observed in cells expressing H2B-GFP-Flag-Chk1 (data not shown). Taking these results together, we conclude that PIKKdependent Chk1 phosphorylation occurs in close proximity of sites of DNA damage and that under normal circumstances, the kinase is then able to dissociate from chromatin, allowing it to spread throughout the nucleus.

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Figure 6. Dissociation of Chk1 from Chromatin Is Required for Effective Arrest of the DNADamage Checkpoint (A) HeLa cells were oligofected with siRNA duplexes directed against Chk1, after which they were transfected with GFP-spectrin expression vector and Flag-Chk1 WT, H2B-Flag-Chk1, or an empty control expression plasmid. Twenty-four hours later, cells were left untreated or were treated with 2 Gy of IR. After 1 hr recovery, cells were harvested, fixed, and stained for MPM-2. MPM-2 positivity of GFPpositive cells was analyzed by FACS analysis. Depicted is the percentage of inhibition of MPM-2-positive cells, compared to untreated control cells, in response to IR treatment. Shown is the average of three independent experiments with error bars representing the standard error. (B) Model for chromatin dissociation of Chk1 in response to genotoxic stress. See text for details.

Dissociation of Chk1 from Chromatin Is Required for a Functional Checkpoint Response To probe the potential functional significance of the dissociation of phosphorylated Chk1 from sites of DNA damage, we examined whether expression of the immobile H2B-Flag-Chk1 fusion protein diminished DNAdamage-induced checkpoint activation. For this, we focused on the IR-induced G2/M checkpoint because this was the key checkpoint defect originally observed in mammalian cells lacking normal Chk1 activity [3, 34], and the key role of Chk1 in this checkpoint has been particularly well documented [4, 23, 35]. Indeed, siRNAmediated downregulation of endogenous Chk1 in HeLa cells efficiently abrogated the IR-induced G2 arrest, and this was restored by expressing Flag-Chk1 in such cells (Figure 6A). By contrast, expression of the immobile H2B-Flag-Chk1 did not restore the checkpoint function in cells that were downregulated for endogenous Chk1 (Figure 6A). These findings indicate that the presence of phosphorylated Chk1 on chromatin (Figures 5A and 5B) is not sufficient for functional DNA-damage signaling and strongly suggest that the chromatin dissociation and mobilization of Chk1 is necessary for efficient DNAdamage checkpoint activation. Discussion While exploring the regulatory mechanisms for Chk1, we found that it binds chromatin and that this association is conserved from mouse to man. Although it is formally possible that Chk1 directly binds to DNA, we think it is more likely that an accessory factor(s) mediates this association. Although proteins already known to regulate Chk1 are strong candidates for such factors, our data show that Hus1, TopBP1, Claspin, and wild-type Nbs1 are not solely required for the Chk1 chromatin association in untreated cells. Furthermore, the association

of ATR with chromatin primarily during S phase [24] suggests that it too does not solely mediate the association of Chk1 with chromatin; and indeed, we have found that downregulation of ATR does not impair chromatin binding by Chk1 (data not shown). It will clearly be of interest to explore what proteins mediate Chk1 chromatin binding. In addition to observing the chromatin association of Chk1, we have established that Chk1 is released from chromatin in the presence of genotoxic stress and that this dissociation strongly correlates with Chk1 phosphorylation. The low affinity of phosphorylated Chk1 for chromatin is consistent with its role as an effector kinase of the DNA-damage response and with the fact that many Chk1 targets are spread throughout the nucleoplasm. Notably, a recent study revealed that, different from Chk1, the mammalian checkpoint effector kinase Chk2 does not stably associate with chromatin [28]. However, immobilizing Chk2 by fusing it to histone H2B nevertheless demonstrated that phosphorylation of Chk2 is restricted to sites of DNA damage caused by laser microirradiation [28], suggesting that Chk2 phosphorylation primarily takes place near sites of damage. By using a similar approach, we showed that phosphorylated Chk1 is found in the chromatin fraction of cells expressing an immobile H2B-Flag-Chk1 fusion, suggesting that Chk1 phosphorylation occurs on the chromatin and that this precedes chromatin dissociation. Moreover, we established that chromatin release is required for the spreading of phosphorylated Chk1 throughout the nucleus, because expression of an immobile form of Chk1 resulted in the restriction of phosphorylated Chk1 to sites of local UV damage. On the basis of these findings, it seems likely that Chk1 and Chk2 must associate with chromatin—specifically in close proximity of the sites of damage—during the early stages of the DNA-damage response in order to be efficiently phosphorylated by

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ATM and ATR, and that Chk1 and Chk2 then dissociate to access their downstream targets. Although Chk1 and Chk2 have overlapping substrate specificities and both seem to be unable to stably associate with chromatin in the presence of genotoxic stress, chromatin binding in unperturbed cells appears to be specific for Chk1. This difference is in agreement with there being key functional differences between these two kinases [3, 34, 36] and might support the hypothesis proposed by Bartek and Lukas that Chk1 operates as a cell-cycle ‘‘workhorse,’’ whereas Chk2 primarily contributes to events that take place following DNA damage [37]. Whereas another group reported the presence of phosphorylated Chk1 on chromatin [21], we have reproducibly found that phosphorylated Chk1 is exclusively present in the soluble fractions as generated by our methods and that phosphorylation of Chk1 seems to regulate its chromatin dissociation. Although we cannot discount the possibility that Chk1 dissociation from the chromatin is in part triggered by modification of an interacting protein, the good correlation we observe between the kinetics and extent of Chk1 phosphorylation and its chromatin dissociation strongly suggests that phosphorylated Chk1 itself has a decreased affinity for chromatin. Our experiments with Chk1 mutated in two PIKK-consensus target sites that are known to undergo DNA-damage-induced phosphorylation lend further support for this idea; however, this mutant still shows some dissociation in response to UV treatment, raising the possibility that additional phosphorylation events contribute to the dissociation of Chk1 from chromatin. Indeed, running SDS-PAGE gels for longer times resulted in a UV-induced mobility shift of the Chk1 S317A/S345A phosphorylation mutant, suggesting that this mutant can undergo additional DNA-damageinduced modifications (Figure S3). Consistent with such a proposal, human Chk1 contains additional SQ motifs, and studies in Xenopus have shown that the equivalent residues in xChk1 can undergo aphidicolin-induced phosphorylation [38]. It will clearly be of interest to determine whether the mutation of these sites influences Chk1 chromatin dissociation and function. What is the functional role of Chk1 dissociation from chromatin? Our data establish that Chk1 is phosphorylated primarily, if not exclusively, on the chromatin flanking sites of DNA damage, which is consistent with it being a DNA-damage PIKK substrate. We have found that only around 20% of Chk1 is associated with chromatin. At low levels of DNA damage, only a small proportion of Chk1 is phosphorylated, so under such circumstances, it could be envisaged that this fraction corresponds to Chk1 that was bound to chromatin before and during the period of DNA damage. By contrast, following exposure to higher doses of damaging agents, the vast majority of cellular Chk1 is phosphorylated in a PIKKdependent manner, which suggests that recruitment of Chk1 molecules from the nucleoplasm to the proximity of the activated PIKKs must have occurred. If phosphorylation of Chk1 reduces its affinity for chromatin, then the dissociation of phosphorylated Chk1 might provide a mechanism to ensure that the phosphorylated, activated Chk1 does not interfere with chromatin recruitment and activation of further Chk1 molecules. Thus, the dissociation of Chk1 from damage sites could

facilitate the phosphorylation and activation of the entire soluble Chk1 pool. Consistent with such model, we found that immobilization of Chk1 inhibits the distribution of phosphorylated Chk1 throughout the nucleus (Figure 5E). Moreover, expression of an immobile H2BFlag-Chk1 fusion protein was unable to rescue the G2/M checkpoint arrest displayed by Chk1-downregulated cells (Figure 6A). Although the primary Chk1 substrates regulating this checkpoint, the Cdc25 phosphatases, are thought to be mobile proteins that shuttle between the nucleus and cytoplasm [39] and might therefore be phosphorylated to some degree by activated, immobilized H2B-Flag-Chk1, our data clearly show that this is not sufficient for a fully functional checkpoint arrest, indicating that dissociation of Chk1 from chromatin contributes to this arrest. Finally, it is possible that the dissociation of Chk1 from chromatin might facilitate the recently reported degradation of Chk1 that takes place in the later stages of the DNA-damage response [40], raising the possibility that it contributes to recovery from checkpoint arrest. Conclusions Our findings point to an additional regulatory mechanism for the DNA-damage-responsive effector kinase Chk1. We show that around 20% of cellular Chk1 is associated with chromatin throughout the cell cycle, which might be required to fulfill its suggested functions during the unperturbed cell cycle. Furthermore, in the presence of genotoxic stress, chromatin binding might serve to localize Chk1 in close proximity to DNA lesions, thereby juxtaposing it with ATM and ATR and facilitating its phosphorylation. Our data show that Chk1 dissociates from chromatin after DNA damage and that this response is closely connected to Chk1 phosphorylation. These findings suggest a model for how Chk1 chromatin association and dissociation contribute to DNA-damage checkpoint events (Figure 6B). Thus, after assembling at sites of damaged DNA, active PIKKs trigger the phosphorylation of chromatin-bound Chk1; this phosphorylation then decreases the affinity of Chk1 for chromatin, leading to a rapid dissociation of Chk1 from the chromatin. In addition to facilitating the chromatin recruitment and activation of further Chk1 molecules, this dissociation allows activated Chk1 to freely move throughout the nucleoplasm in order to phosphorylate cell-cycle regulators and thereby efficiently control and coordinate various downstream events of the DNA-damage response. Experimental Procedures Cell Lines, Reagents, and Treatments U2OS and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine. Hus1+/+p212/2 and Hus12/2p212/2 MEFs were a gift from R.S. Weiss (Cornell University, New York) and were grown on gelatincoated dishes, in the medium described above. The genotype of these cells was confirmed with PCR and western blotting. Caffeine, deoxycytidine, thymidine, hydroxyurea, nocodazole, and micrococcal nuclease were obtained from Sigma. Wortmannin was from Alexis Biochemicals. KU-55933 was kindly provided by G.C. Smith (KuDOS Pharmaceuticals, Cambridge, United Kingdom). Flag-agarose beads were from Sigma. Antibodies used in this study were obtained from Abcam (Chk2, Claspin, and TopBP1), BD

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Transduction (Grb2), Cell Signaling (pS345-Chk1 and pS317-Chk1), Chemicon (Actin), BD PharMingen (Orc2), Santa Cruz Biotechnology (Chk1), Sigma (Flag), and Upstate Biotechnology (Chk1 and MPM-2). Anti-p62 was a gift from J.M. Egly (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France). U2OS cells were synchronized at the G1/S transition by a double thymidine block. To this end, cells were treated with 2.5 mM thymidine for 24 hr, after which the cells were released from the block by washing twice with PBS. Twelve hours after the release, thymidine was added for another 20 hr. Then, cells were released from the second thymidine block by washing twice with PBS and adding fresh medium with 24 mM deoxycytidine. Four hours after the release, nocodazole (250 ng/ml) was added to the cells, to prevent cells from progressing into G1 phase. At different time points after release from the second thymidine block, cells were harvested by trypsinization. Cells were synchronized at the metaphase/anaphase transition by treatment with 250 ng/ml nocodazole for 16 hr. To release detached cells from mitosis, we washed them with PBS and reseeded them. At different time points after release, cells were harvested by trypsinization. After trypsinization, part of the cells was fixed in 70% ethanol to check cell-cycle stage by FACS analysis, and the other part was used for chromatin fractionation. Cells were treated with IR by using a 137Cs source.

Plasmids, Oligonucleotides, and Transfections pCIneo Flag-Chk1 WT (wild-type), pCIneo Flag-Chk1 S317A/S345A, and pCIneo Flag-Chk1 D130A were kindly provided by J. Bartek (Institute of Cancer Biology, Copenhagen, Denmark). GFP, H2B, and H2B-GFP ORFs were amplified by PCR from pEF-Bos-H2B-GFP [31] and inserted in-frame into pCIneo Flag-Chk1 to generate pCIneo GFP-Flag-Chk1, pCIneo H2B-Flag-Chk1, and pCIneo H2B-GFP-FlagChk1, respectively. pPCMV eGFP-spectrin has been described [41]. The luciferase (CGUACGCGGAAUACUUCGAdTdT), Chk1 (UCGU GAGCGUUU GUUGAACdTdT), TopBP1 (GUGGUUGUAACAGCGCA UCdTdT), and Claspin (CCUUGCUUAGAGCUGAGUCdTdT) siRNA duplexes were obtained from Dharmacon Research. Transient transfections of plasmids were carried out with the standard calcium-phosphate transfection protocol. Transfection of siRNA was performed with Oligofectamine (Invitrogen). The final concentration of the siRNA duplex in the culture medium was 100 nM and was left on the cells for 48 or 72 hr.

Whole-Cell Extracts and Chromatin Fractionation For WCE, cells were washed in cold PBS, after which the cells were resuspended in Laemmli sample buffer (4% SDS, 20% glycerol, 120 mM Tris [pH 6.8]). Laemmli extracts were then boiled for 5 min and sheared by syringing through a 25G needle. Chromatin fractionation of mammalian cells was performed essentially as described [8, 22]. Approximately 3 3 106 cells were washed in PBS and resuspended in 200 ml of solution A (10 mM Hepes [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, protease and phosphatase inhibitors). Triton X-100 was added to a final concentration of 0.1%, cells were incubated on ice for 5 min, and the cytoplasmic (S1) and nuclear fractions (P1) were harvested by centrifugation at 1,300 3 g for 4 min. Isolated nuclei were then washed in solution A, lysed in 150 ml solution B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease and phosphatase inhibitors), and incubated on ice for 10 min. The soluble nuclear (S2) and chromatin fractions were harvested by centrifugation at 1,700 3 g for 4 min. Isolated chromatin (P2) was then washed in solution B, spun down at 10,000 3 g, and resuspended in 150 ml Laemmli sample buffer. Where indicated, fractions S1 and S2 were pooled to one soluble fraction. To release chromatin-bound proteins by nuclease treatment, we resuspended cell nuclei (P1) in buffer A plus 1 mM CaCl2 and 2 U micrococcal nuclease for 2 min at 37ºC, after which the reaction was stopped by the addition of 1 mM EGTA. Nuclei were collected by centrifugation at 1,300 3 g for 4 min and lysed as described above. Protein concentrations were determined with the Lowry protein assay, and equal amounts of protein were loaded onto SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked, incubated with the indicated antibodies, and developed with enhanced chemiluminescence.

Immunofluorescence Cells were grown on glass coverslips. Local UV damage was induced with 5 mm isopore polycarbonate membrane filters (Millipore) as previously described [32]. After 1 hr, cells were washed with PBS and subsequently fixed and permeabilized with 2% paraformaldehyde containing 0.2% Triton X-100 for 20 min. Samples were blocked in 3% BSA and immunostained with antibodies as indicated. Fluorescence images were captured and processed with a Laser Scanning Microscope and LSM Software (Zeiss). G2/M Checkpoint Assay HeLa cells were transfected with Chk1 siRNA oligos. Forty-eight hours later, cells were transfected with 10 mg of pCIneo Flag-Chk1 WT or pCIneo H2B-Flag-Chk1 and 1 mg of GFP-spectrin expression vector. Even though exogenous (H2B-)Flag-Chk1 levels were also somewhat downregulated by Chk1 siRNA, 24 hr after transfection, the levels of exogenous (H2B-)Flag-Chk1 were similar to the levels of endogenous Chk1 in control cells that had been transfected with Luciferase siRNA oligos. At this time, the endogenous Chk1 was undetectable by western blot in cells transfected with Chk1 siRNA oligos (data not shown). Cells were irradiated with 2 Gy of IR and incubated for 1 hr at 37ºC. Subsequently, they were collected by trypsinization and fixed in 70% ethanol overnight at 4ºC. After fixation, cells were washed with PBS and incubated with an antibody that recognizes the mitotic epitope MPM-2 for 1 hr at 4ºC. After they were washed with PBS, cells were incubated with a Cy5-labeled secondary antibody (Jackson Immunoresearch) for 30 min at 4ºC, followed by staining the DNA with propidium iodide. MPM-2 positivity of GFP-positive cells was analyzed by flow cytometry with FACSCaliber and CellQuest Pro software (Becton Dickinson). Supplemental Data Supplemental Data include three figures and are available with this article online at: http://www.current-biology.com/cgi/content/full/ 16/2/150/DC1/. Acknowledgments We thank Robert Weiss for the Hus1 null MEFs, Jiri Bartek for the Chk1 plasmids, Jean-Marc Egly for the p62 antibody, and Graeme Smith for the ATM inhibitor. We also thank the members of Jackson lab for helpful discussions and advice; Jacob Falck, Ali Jazayeri, and Raimundo Freire for critical reading of the manuscript; and Roland Kanaar and members of the Genetics Department for support. Research in the Jackson laboratory is supported by Cancer Research UK, with core infrastructure funding being provided by Cancer Research UK and the Wellcome Trust. P.M.R. was funded by a Cancer Research UK studentship. V.A.J.S. is supported by the Dutch Cancer Society and the Association for International Cancer Research. Received: July 5, 2005 Revised: November 22, 2005 Accepted: November 24, 2005 Published online: December 15, 2005 References 1. Abraham, R.T. (2001). Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15, 2177–2196. 2. Matsuoka, S., Huang, M., and Elledge, S.J. (1998). Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282, 1893–1897. 3. Liu, Q., Guntuku, S., Cui, X.S., Matsuoka, S., Cortez, D., Tamai, K., Luo, G., Carattini-Rivera, S., DeMayo, F., Bradley, A., et al. (2000). Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev. 14, 1448–1459. 4. Gatei, M., Sloper, K., Sorensen, C., Syljuasen, R., Falck, J., Hobson, K., Savage, K., Lukas, J., Zhou, B.B., Bartek, J., et al. (2003). Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation. J. Biol. Chem. 278, 14806–14811.

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