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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Mol Cell. 2008 Dec 26;32(6):862–869. doi: 10.1016/j.molcel.2008.12.005

The role of Dbf4/Drf1-dependent kinase Cdc7 (Ddk) in DNA damage checkpoint control

Toshiya Tsuji 1, Eric Lau 1,2, Gary G Chiang 1, Wei Jiang 1,3
PMCID: PMC4556649  NIHMSID: NIHMS85020  PMID: 19111665

Summary

The Dbf4/Drf1-dependent S-phase promoting kinase Cdc7 (Ddk) is thought to be an essential target inactivated by the S-phase checkpoint machinery that inhibits DNA replication. However, we show here that the complex formation, chromatin-association, and kinase activity of Ddk are not inhibited during the DNA damage-induced S-phase checkpoint response in Xenopus egg extracts and mammalian cells. Instead, we find that Ddk plays an active role in regulating S-phase checkpoint signaling. Addition of purified Ddk to Xenopus egg extracts or overexpression of Dbf4 in HeLa cells downregulates ATR-Chk1 checkpoint signaling and overrides the inhibition of DNA replication and cell cycle progression induced by DNA damaging agents. These results indicate that Ddk functions as an upstream regulator to monitor S-phase checkpoint signaling. We propose that Ddk modulates the S-phase checkpoint control by attenuating checkpoint signaling and triggering DNA replication re-initiation during the S-phase checkpoint recovery.

Keywords: Ddk, DNA replication, S-phase checkpoint

Introduction

In all eukaryotes, the initiation of DNA replication is controlled by the stepwise establishment of pre-replication complexes (pre-RCs) at DNA replication origins in G1 (replication licensing) and the activation of pre-RCs by two S-phase promoting kinases, cyclin-dependent kinases (Cdks) and Dbf4/Drf1-dependent kinase Cdc7 (Ddk) in G1/S-S (Bell and Dutta, 2002; Sclafani and Holzen, 2007). Aberrant DNA replication and DNA damage in S-phase immediately lead to activation of the S-phase checkpoint, which suppresses late origin firing to prevent further DNA replication and stabilizes stalled replication forks to ensure proper fork restart following removal of the replication error. It is well known that DNA damage or stalled replication forks activate the checkpoint kinases, ATM (ataxia-telangiectasia-mutated)-Chk2 and ATR (ATM and Rad3-related)-Chk1. While the ATM-Chk2 signaling pathway usually responds to double-stranded DNA breaks, the ATR-Chk1 signaling pathway is activated by a broad spectrum of DNA lesions and replication blocks. The molecular mechanisms by which the S-phase checkpoint is activated by DNA damages/lesions have been extensively studied (Yang and Zou, 2006). However, less is known how the S-phase checkpoint is attenuated once DNA damages/lesions are repaired (Freire et al., 2006; Gewurz and Harper, 2006).

The requirement of Ddk kinase activity for DNA replication suggests that Ddk plays an important role in S-phase checkpoint control (Jares et al., 2000). However, disparate results were obtained from different studies in various systems, and even within the same system. Some studies suggested that Ddk is a final target inactivated by the S-phase checkpoint response whereas other studies suggested that Ddk plays an active role in regulating the S-phase checkpoint response (Costanzo et al., 2003; Dierov et al., 2004; Heffernan et al., 2007; Liu et al., 2006; Petersen et al., 2006; Silva et al., 2006; Tenca et al., 2007). For instance, in the Xenopus cell-free DNA replication system, Ddk activity was shown to be inhibited through the dissociation of the Cdc7/Dbf4 complex after egg extracts were treated with etoposide (ETO), a DNA topoisomerase II inhibitor that causes single-strand breaks and activates the ATR-dependent S-phase checkpoint (Costanzo et al., 2003). Dissociation of the Cdc7/Dbf4 complex was also reported in Bcr-Abl negative, ATR-proficient human leukemia cells treated with ETO, supporting the notion that Cdk is an essential target of the S-phase checkpoint (Dierov et al., 2004). However, recent studies indicated that Cdc7/Drf1, not Cdc7/Dbf4, is the major form of Ddk in Xenopus egg extracts, calling the previous Xenopus study into question (Silva et al., 2006; Takahashi and Walter, 2005). Furthermore, following exposure to ETO, Ddk (Cdc7/Drf1 or Cdc7/Dbf4) kinase activity is unaffected in Xenopus egg extracts and mammalian cells, including Bcr-Abl negative, ATR-proficient human leukemia cells, which supports the possibility that Ddk is not a final target of the S-phase checkpoint, although the precise role of Ddk in the S-phase checkpoint was not elucidated in these studies (Heffernan et al., 2007; Liu et al., 2006; Petersen et al., 2006; Silva et al., 2006; Tenca et al., 2007). In this study, we show that Ddk functions as an upstream regulator to monitor the S-phase checkpoint response, suggesting that Ddk is actively involved in S-phase checkpoint control by attenuating checkpoint signaling and triggering DNA replication re-initiation during the S-phase checkpoint recovery.

Results and Discussion

The role of Ddk in S-phase checkpoint response in Xenopus egg extracts

To determine the precise role of Ddk in S-phase checkpoint response in higher eukaryotes, we generated and purified active and fully functional recombinant human Cdc7/Dbf4 or Cdc7/Drf1 and Cdc7kd/Dbf4 (kinase dead) or Cdc7kd/Drf1 (kinase dead) complexes from Sf9 cells using a baculovirus expression system (Figure S1). The effects of Ddk on S-phase checkpoint response induced by DNA damaging agents were examined in Xenopus egg extracts. Consistent with previous reports (Costanzo et al., 2003), DNA replication was greatly inhibited when egg extracts were treated with more than 30 μM ETO (Figure S2A). Such inhibition could be blocked by caffeine, a nonspecific inhibitor of ATR/ATM (Figure S2B), indicating that the inhibition is S-phase checkpoint dependent. Addition of purified Ddk (Cdc7/Dbf4, Cdc7/Drf1 or Cdc7/Dbf4 and Cdc7/Drf1 together) could also restore DNA synthesis in ETO-treated extracts in a dose-dependent manner and such restoration was blocked by an excess amount of ETO (300 μM) (Figure 1A, 1B and S2B). Similar results were also obtained from egg extracts treated with other DNA damaging agents, such as camptothecin (CPT) or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (Figure 1B). Thus, these results indicate that Ddk is involved in S-phase checkpoint control in egg extracts.

Figure 1.

Figure 1

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Restoration of DNA replication by Ddk (Cdc7/Dbf4, Cdc7/Drf1 or Cdc7/Dbf4 and Cdc7/Drf1 together) in DNA damaging agent treated egg extracts. Egg extracts supplemented with [α-32P]dCTP were treated without or with 30 μM or 300 μM ETO (A and B), 30 μM CPT (B) or 30 μM MNNG (B) in the absence or presence of purified Ddk at the indicated concentrations and incubated with Xenopus sperm chromatin for 90 min. After incubation, the reactions were subjected to agarose gel electrophoresis and DNA synthesis was measured by autoradiography using PhosphorImager.

On the basis of these results, two possible models for the action of Ddk in S-phase checkpoint control could be proposed. The first model is that the S-phase checkpoint inactivates Ddk to block the initiation of DNA replication (Costanzo et al., 2003). In this scenario, addition of active exogenous Ddk to egg extracts can overcome the inhibition of endogenous Ddk induced by S-phase checkpoint signaling and promote DNA replication. The second model is that Ddk is not a final downstream target of the S-phase checkpoint (Silva et al., 2006; Tenca et al., 2007) but instead plays an active role as an upstream regulator to modulate S-phase checkpoint signaling. In the latter scenario, addition of active Ddk would attenuate the S-phase checkpoint response and trigger DNA replication re-initiation.

To distinguish between these two models and determine at what point Ddk acts, we examined the chromatin association, complex formation and kinase activities of endogenous and exogenous Ddks and their effects on DNA replication in ETO-treated extracts. Immunoblotting analysis of egg extracts treated with ETO and incubated with sperm chromatin revealed that, regardless of DNA replication status, the association of endogenous XCdc7, XDbf4 or XDrf1 and exogenous Dbf4 or Drf1 on chromatin was not affected by ETO treatment, even at 300 μM ETO (Figure 2Aa–d and g). These results indicate that the chromatin association of Cdc7 as well as Dbf4 and Drf1 is not regulated by the ETO-induced S-phase checkpoint (Silva et al., 2006; Yanow et al., 2003). Immunoprecipitation analysis showed that exogenous Dbf4 or Drf1 protein not only associated with exogenous Cdc7, which was present in the added complex, but also could interact with endogenous XCdc7 in the extracts, presumably by inter-molecular exchange (Figure 2Ba–c). These results indicated that ETO treatment of extracts did not abolish Cdc7/Dbf4 or Cdc7/Drf1 complex formation, a result that contrasts with previous reports (Costanzo et al., 2003). In vitro kinase assays using recombinant MCM2 as a substrate indicated that exogenous nucleosolic Cdc7/Dbf4 or Cdc7/Drf1 and endogenous nucleosolic or chromatin-bound XCdc7 kinase activities were also not affected by ETO treatment (Figure 2Bd and 2C). Thus, the unaltered chromatin association, complex formation and kinase activities of endogenous and exogenous Ddks during ETO-induced S-phase checkpoint response demonstrated that Ddk inactivation by the S-phase checkpoint is not the mechanism by which DNA replication is blocked in egg extracts.

Figure 2.

Figure 2

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ETO treatment does not affect Ddk complex formation, chromatin association or kinase activity in egg extracts. (A) Egg extracts treated without or with 30 μM or 300 μM ETO in the absence or presence of 150 nM purified Ddk (Cdc7/Dbf4 or Cdc7/Drf1) were incubated with Xenopus sperm chromatin for 90 min. After incubation, sperm chromatin was extracted and isolated by centrifugation. Chromatin pellets were subjected to SDS/PAGE, transferred to PVDF membrane and immunoblotted with anti-XCdc7 (a), anti-XDbf4 (b), anti-XDrf1 (c), anti-FLAG (d), anti-XMCM10 (e) or anti-XCdc45 antibodies (f). In parallel, extracts treated under the same conditions were supplemented with [α-32P]dCTP and incubated with Xenopus sperm chromatin for 90 min. After incubation, the reactions were subjected to agarose gel electrophoresis and DNA synthesis was determined by autoradiography (g). (B) Egg extracts treated as in (A) were immunoprecipitated using anti-FLAG M2 monoclonal antibody. The immunoprecipitates were either subjected to SDS/PAGE, followed by immunoblotting with anti-FLAG (a), anti-Cdc7 (b) or anti-XCdc7 antibodies (c), or incubated with purified N-terminal region of MCM2 (MCM2M5, residues 1-178) in the presence of [γ-32P]ATP for kinase assays. The kinase reactions were resolved by SDS/PAGE, followed by autoradiography (d). (C) Egg extracts were treated as in (A) and the formed nuclei were isolated and extracted by CSK/0.5% TritonX100 buffer. Nuclear soluble fraction (a) or nuclear insoluble fraction solubilized by DNase I treatment (b) was immunoprecipitated using anti-XCdc7 antibodies. The immunoprecipitates were incubated with MCM2M5 as a substrate in the presence of [γ-32P]ATP for kinase assays. The kinase reactions were analyzed by autoradiography following SDS/PAGE separation.

How then does the ETO-induced S-phase checkpoint inhibit the initiation of DNA replication and how does addition of active Ddk override such inhibition? To answer these questions, we examined the chromatin-association of other DNA replication factors in egg extracts in the presence or absence of ETO and exogenous Cdc7/Dbf4 or Cdc7/Drf1. We found that the chromatin association of two DNA replication initiators, XMCM10 or XCdc45, tightly correlated with the DNA replication capacity of the extracts (Figure 2Ae–g). Since the chromatin loading of MCM10 and Cdc45 are crucial for the initiation of DNA replication (Wohlschlegel et al., 2002; Zou and Stillman, 1998), our results indicated that inhibiting the loading of XMCM10 and XCdc45 onto chromatin by the ETO-induced S-phase checkpoint signaling blocked the initiation of DNA replication. However, this inhibition can be overridden by the addition of active Ddk, which restored DNA replication. Thus, addition of Ddk could override the inhibition of DNA replication by the ETO- (or other DNA damaging agents) induced S-phase checkpoint, strongly indicating that Ddk plays an active role, such as an upstream regulator, in regulating S-phase checkpoint signaling.

The role of Ddk in S-phase checkpoint response in mammalian cells

To determine whether Ddk also plays a similar role in DNA damage checkpoint control in mammalian cells, we generated two HeLa cell lines (56α and 57α) that stably overexpress human FLAG-tagged Dbf4 under the control of a tetracycline (Tet) inducible system. Immunoblotting analysis and kinase assays showed that the expression levels of exogenous FLAG-Dbf4 and Dbf4-associated Cdc7 kinase activity increased dramatically upon induction of Dbf4 expression in these cells (Figure S3A). Using FACS analysis, we examined the cell cycle profiles of 56α or 57α cells in the presence or absence of 0.5 μM ETO in concert with uninduced or induced Dbf4 expression. This concentration of ETO (0.5 μM) was chosen because it induced significant cell cycle arrest without causing dramatic cell death in these cells (Figure S3B). In the absence of ETO, induction of Dbf4 expression did not appreciably alter the cell cycle profile of 56α or 57α cells, indicating that Dbf4 overexpression does not perturb cell cycle progression (Figure 3A). However, in the presence of ETO alone, 56α or 57α cells blocked in the S-G2/M phase, indicating that ETO treatment activated the DNA damage checkpoint and inhibited cell cycle progression (Cliby et al., 2002). In contrast, in the presence of both Dbf4 overexpression and ETO, the number of 56α or 57α cells in the S-G2/M phase was reduced but an increased population of sub-G1 phase cells was observed (Figure 3A and S3C). Thus, like our previous experiments in Xenopus egg extracts, induced overexpression of Dbf4 in mammalian cells overrides the ETO-induced DNA damage checkpoint, releasing cells into the cell cycle and ultimately resulting in cell death.

Figure 3.

Figure 3

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Effects of ETO treatment on cell cycle progression, Ddk expression, chromatin association and kinase activity in 56α and 57α cells overexpressing Dbf4. (A) Tetracycline-inducible (Tet-off) HeLa cell lines, 56α and 57α overexpressing Dbf4 cultured in the presence (Dbf4 uninduced) or absence (Dbf4 induced) of tetracycline for 48 h were treated without or with 0.5 μM ETO for the indicated times (h). The cell cycle parameters were then determined by FACS analysis. Schematic experimental procedures are also shown. (B and C) 56α and 57α cells cultured for 48 h in the presence (lanes 1, 3, 5 and 7) or absence (lanes 2, 4, 6 and 8) of tetracycline were treated without (lanes 1, 2, 5, and 6) or with (lanes 3, 4, 7 and 8) 0.5 μM ETO for 24h. (B) Cells were lysed in 1% NP-40 buffer and 10% of cell lysates were subjected to SDS-PAGE followed by immunoblotting with anti-FLAG (a), anti-Dbf4 (b) or anti-Cdc7 antibodies (c). The remaining lysate was immunoprecipitated with anti-Cdc7 antibodies. The immunoprecipitates were used for Cdc7 kinase assays with the purified N-terminal region of MCM2 (MCM2M5) as a substrate in the presence of [γ-32P]ATP. After incubation, the kinase reactions were resolved by SDS/PAGE, followed by autoradiography (d). (C) Cells were extracted with CSK/0.5% TritonX-100 buffer. After washing, the insoluble cell fraction was lysed in sample buffer. After sonication, cell lysates were separated by SDS/PAGE and immunoblotted with the indicated antibodies. Densitometric quantitation of Cdc7, Dbf4 or MCM2S139P levels normalized with control α-tubulin or total MCM2 levels was performed using image J software (NIH).

We determined the effects of ETO treatment on Cdc7/Dbf4 expression, chromatin association, complex formation and kinase activity (measured by an in vitro kinase assay or by immunoblotting for MCM2 phosphorylation with a phospho-Ser139 specific antibody (Tsuji et al., 2006)) in 56α or 57α cells under induced and uninduced expression conditions. Immunoblotting, coimmunoprecipitation and kinase assays indicated that ETO treatment did not reduce Cdc7 and Dbf4 (exogenous/endogenous) expression, their chromatin association and complex formation, or Cdc7/Dbf4 kinase activity in 56α or 57α cells under the uninduced or induced conditions (Figure 3B, 3C and Figure S4A). Instead, ETO treatment resulted in increased chromatin-association of Cdc7/Dbf4 and Cdc7/Dbf4-dependent phosphorylation of MCM2 (Figure 3C, and S4B), consistent with recent reports (Tenca et al., 2007). Thus, similar to what we observed in Xenopus egg extracts (Figure 2), these results demonstrated that Ddk is not a final target of ETO-induced checkpoint response in mammalian cells. As Ddk accumulated on the chromatin after ETO treatment and overexpression of Dbf4 overrode the ETO-induced cell cycle block and ultimately caused cell death in HeLa cells, the results also highlight that Cdc7 kinase functions as an upstream regulator in regulating the DNA damage-induced S-phase checkpoint control in mammalian cells.

Ddk is involved in downregulating the S-phase checkpoint signaling

To better understand the mechanism by which Ddk regulates the DNA damage-induced S-phase checkpoint pathway, we investigated the status of the ATR-Chk1 checkpoint signaling pathway in Xenopus egg extracts and mammalian cells in response to ETO and elevated Ddk levels. In egg extracts, immunoblotting analysis of Chk1 phosphorylation at Ser344, an indicator of ATR-dependent Chk1 activation (Zhao and Piwnica-Worms, 2001), detected dramatically increased levels of Chk1 phosphorylation upon treatment with 30 μM or 300 μM ETO (Figure 4A). Chk1 phosphorylation coincided with the inhibition of DNA replication, confirming that ETO treatment activated the ATR-Chk1 checkpoint. Addition of purified Cdc7/Dbf4 (150 nM) to egg extracts treated with 30 μM ETO significantly reduced Chk1 phosphorylation levels and was coincident with restoration of DNA replication. Time course and titration experiments further confirmed the results (Figure 4B–4C), although addition of purified Cdc7/Dbf4 (150 nM) did not reduce the levels of Chk1 phosphorylation in egg extracts treated with 300 μM ETO and was unable to restore DNA replication. In mammalian cells, similar effects on the phosphorylation of Chk1 and other ATR-downstream effectors such as Cdc2 (Tyr15) and histone H3 (Ser10) were observed in 56α and 57α cells overexpressing Dbf4 (Figure 4D). As Ddk promotes replication initiation, it was formally possible that the inhibition of checkpoint activation observed in the presence of Ddk was an indirect effect resulting from an increased firing of replication origins and not due to a direct effect of Ddk on the S-phase checkpoint machinery. In order to rule out this possibility, egg extracts in mid-S phase were treated with the Cdk inhibitor p27, which inhibited all new initiation events. Similar to what we observed in the absence of p27, ETO treatment in egg extracts in the presence of p27 also resulted in an increase of Chk1 phosphorylation and this increase could be inhibited by addition of purified Cdc7/Dbf4 (Figure 5A). These results indicated that inhibition of checkpoint activation was a direct rather than indirect effect of Ddk to promote replication initiation. Taken together, our results demonstrate that Ddk can override S-phase checkpoint control by downregulating ATR-Chk1 checkpoint signaling.

Figure 4.

Figure 4

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Inhibition of Chk1 phosphorylation by Ddk (Cdc7/Dbf4) in ETO untreated or treated egg extracts and mammalian cells. (A) Egg extracts untreated (lanes 1 and 2) or treated with ETO (30 μM, lanes 3 and 4, 300 μM, lanes 5 and 6) in the absence (lanes 1, 3 and 5) or presence (lanes 2, 4 and 6) of 150 nM purified Cdc7/Dbf4 were incubated with Xenopus sperm chromatin for 90 min. After incubation, nuclei were isolated and lysed with sample buffer. Lysates were separated by SDS/PAGE followed by immunoblotting with anti-phospho-Chk1S344 (a), anti-Chk1 (b) or anti-XOrc2 antibodies (c, loading control). Densitometric quantitation of Chk1S344P levels normalized with control Orc2 levels was carried out using image J software (NIH). In parallel, extracts treated under the same conditions were supplemented with [α-32P]dCTP and incubated with Xenopus sperm chromatin for 90 min. After incubation, the reactions were subjected to agarose gel electrophoresis and DNA synthesis was determined by autoradiography (d). (B) Egg extracts treated with buffer only (lanes 1–6) or 150 nM purified Cdc7/Dbf4 (lanes 7, 8 and 9) were incubated with Xenopus sperm chromatin in the absence (lanes 1, 2 and 3) or presence of 30 μM ETO (lanes 4–9). After the indicated time points (60 min: lanes 1, 4, and 7; 90 min: lanes 2, 5, and 8; 120 min: lanes 3, 6, and 9), nuclei were isolated by centrifugation onto a sucrose-cushion. After washing, the collected nuclei were lysed with sample buffer and then subjected to SDS/PAGE followed by immunoblotting with anti-phospho-Chk1S344 (a), anti-Chk1 (b) or anti-XOrc2 antibodies (c, loading control). Densitometric quantitation of Chk1S344P levels normalized with control Orc2 levels was performed using image J software (NIH). (C) Egg extracts treated with buffer only (lanes 1 and 2) or purified Cdc7/Dbf4 at different concentrations (lane 3: 1.2 nM, lane 4: 6 nM, lane 5: 30 nM, lane 6: 150 nM) were incubated with Xenopus sperm chromatin for 90 min in the absence (lane 1) or presence of 30 μM ETO (lanes 2–6). After the incubation, nuclei were isolated by centrifugation onto a sucrose-cushion, washed, lysed with sample buffer, and subjected to SDS-PAGE followed by immunoblotting with anti-phospho-Chk1S344 (a), anti-Chk1 (b) or anti-XOrc2 antibodies (c, loading control). Densitometric quantitation of Chk1S344P levels normalized with control Orc2 levels was carried out using image J software (NIH). (D) 56α and 57α cells cultured for 48h in the presence (lanes 1, 3, 5 and 7) or absence (lanes 2, 4, 6 and 8) of tetracycline were treated without (lanes 1, 2, 5 and 6) or with 0.5 μM ETO (lanes 3, 4, 7 and 8) for an additional 24 h. Cells were lysed in 1% NP40 buffer as in Figure S4C and cell lysates were subjected to SDS/PAGE followed by immunoblotting with anti-Chk1 (a, top), anti-phospho-Chk1S345 (a, bottom), anti-histone H3 (b, top), anti-phospho-histone H3S10 (b, bottom), anti-Cdc2 (c, top) or anti-phospho-Cdc2Y15 (c, bottom) antibodies, respectively. Densitometric quantitation of Chk1S345P, Histone H3P or Cdc2Y15P levels normalized with total Chk1, Histone H3 or Cdc2 levels was performed using image J software (NIH).

Figure 5.

Figure 5

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Effects of ETO on Chk1 phosphorylation in the presence of purified recombinant Cdk inhibitor, p27, and Ddk in Xenopus egg extracts. (A) (a) Egg extracts incubated with sperm chromatin in the presence of [α-32P]dCTP were treated with 1 μM purified recombinant GST-p27 at the indicated time. After incubating for a total of 90 min, the reactions were subjected to agarose gel electrophoresis and DNA synthesis was measured by autoradiography. (b) A schematic experimental procedure of egg extracts that were incubated with sperm chromatin in the absence or presence of 30 μM ETO, 1 μM purified recombinant GST-p27 and 150 nM purified Cdc7/Dbf4 at the indicated time. (c) Following incubation, nuclei from egg extracts shown in (b) were isolated by centrifugation onto a sucrose-cushion. After washing, the collected nuclei were lysed with sample buffer and subjected to SDS-PAGE followed by immunoblotting with the indicated anti-phospho-Chk1S345, anti-Chk1 or anti-XOrc2 antibodies. Densitometric quantitation of Chk1S344P levels normalized with control Orc2 levels was performed using image J software (NIH). (B) A schematic model for the involvement of Ddk in regulating the initiation of DNA replication and the S-phase DNA replication/DNA damage checkpoint (for details, see text).

In this study, we provide compelling evidence that Ddk is not an essential target that is inactivated by the S-phase checkpoint to block DNA replication, but rather plays an active role in regulating S-phase checkpoint signaling. Previously, it was shown that DNA lesions generated by ETO treatment activated the ATR-Chk1 checkpoint that blocks the initiation of DNA replication (Costanzo et al., 2003; Dierov et al., 2004). This block in DNA synthesis was attributed to an inhibition of Cdc7/Dbf4 complex formation and kinase activity, resulting in the subsequent failure of Cdc45 loading onto chromatin. Therefore, it was proposed that Ddk is an essential target of the ATR-Chk1 checkpoint that blocks the initiation of DNA replication. However, recent studies suggested that the Ddk complex (Cdc7/Drf1 or Cdc7/Dbf4) is stable and active in higher eukaryotes following DNA damage (Heffernan et al., 2007; Liu et al., 2006; Silva et al., 2006; Takahashi and Walter, 2005; Tenca et al., 2007). Consistent with these recent findings, our results demonstrate that the expression, complex formation, chromatin-association and kinase activity of Ddk are not perturbed during ETO-induced S-phase checkpoint response in both Xenopus egg extracts and mammalian cells, strongly arguing that Ddk is not a final target for inactivation by the S-phase checkpoint to block DNA replication.

Although Ddk is not a target of the S-phase checkpoint, our results show that ETO treatment results in the accumulation of chromatin-associated Cdc7/Dbf4 in mammalian cells, consistent with previous reports (Tenca et al., 2007). Moreover, addition of purified active Ddk to Xenopus egg extracts or overexpression of Dbf4 in mammalian cells could downregulate the ATR-Chk1 checkpoint signaling and override the inhibition of DNA replication initiation and cell cycle progression induced by ETO (or CPT or MNNG), indicating that Ddk is involved in regulating the S-phase checkpoint by abrogating checkpoint signaling. The target(s) of Ddk in the S-phase checkpoint signaling pathway remains to be determined. However, one might expect that Ddk could directly target the S-phase checkpoint sensors or transducers, such as ATR/ATRIP/TopBP1, Rad17-RFC, the 9-1-1 complex, Chk1/Claspin/timeless to attenuate the checkpoint signal (Yang and Zou, 2006). Alternatively, Ddk could activate the S-phase checkpoint recovery pathway to turn off the checkpoint (Freire et al., 2006; Gewurz and Harper, 2006).

Our results also show that inhibition of the chromatin loading of MCM10 and Cdc45 coincides with the inhibition of DNA replication by the S-phase checkpoint, indicating that such inhibition is a critical step to block DNA synthesis by the S-phase checkpoint response. Consistent with our results, several previous studies in various systems showed that the chromatin loading of Cdc45 was inhibited during S-phase checkpoint activation induced by genotoxic stress (Aparicio et al., 1997; Liu et al., 2006; Yanow et al., 2003). Since the chromatin loading of MCM10 is independent of Ddk activity and required for the chromatin loading of Cdc45 (Wohlschlegel et al., 2002), our results suggest that inhibition of the chromatin loading of MCM10 could be a key early step for inhibition of DNA replication by the S-phase checkpoint.

Based on our results and current understanding of the initiation of DNA replication and S-phase checkpoint control, we propose the following model for Ddk regulation of these processes in higher eukaryotes (Figure 5B). During G1/S, activation of Ddk, due to increased expression of Dbf4/Drf1, results in phosphorylation of chromatin-bound MCM proteins and the stable chromatin association of Ddk (Sheu and Stillman, 2006; Tsuji et al., 2006). Chromatin-bound MCM and Ddk are required for the chromatin loading of Cdc45 and GINS, presumably promoting the formation of the Cdc45/MCM/GINS (CMG) complex and activation of CMG DNA replicative helicase activity (Moyer et al., 2006; Sheu and Stillman, 2006; Tsuji et al., 2006). Chromatin loading of Cdc45 also requires the chromatin association of MCM10 (Wohlschlegel et al., 2002). The CMG complex formation and activation are essential for the initiation of DNA replication. During genotoxic stress, the S-phase DNA replication/damage checkpoint is activated. The S-phase checkpoint signaling pathway targets MCM10 to block its chromatin association, which subsequently abrogates the chromatin loading of Cdc45, thus inhibiting formation of the CMG complex, activation of the DNA replicative helicase and ultimately the initiation of DNA replication. However, because the S-phase checkpoint signaling pathway does not target Ddk for inactivation on the chromatin, the complex remains chromatin-associated and is able to phosphorylate the chromatin-associated MCM complexes. During the damage recovery interval, Ddk accumulates on the chromatin resulting in the Ddk-dependent down-regulation of the S-phase checkpoint. Attenuation of the S-phase checkpoint signaling by Ddk thus allows the chromatin reloading of MCM10, which together with Ddk, promotes the chromatin reloading of Cdc45 and GINS to reform the CMG complex with MCM proteins. The formation and activation of the CMG DNA replicative helicase ultimately triggers the re-initiation of DNA replication.

Experimental Procedures

Plasmids, baculoviruses and antibodies

The coding sequences of human FLAG-tagged Dbf4, FLAG-tagged Drf1, Myc-tagged Cdc7 and Myc-tagged Cdc7kd (kinase dead, D196N substitution) were amplified by PCR and subcloned into the baculovirus vector pFastBacHTa (Invitrogen). The coding sequence of human FLAG tagged Dbf4 was amplified by PCR and subcloned into the Tet-off mammalian expression vector pTRE2puro (BD Biosciences). All constructs were confirmed by DNA sequencing. The baculoviruses that express Dbf4, Drf1, Cdc7, or Cdc7kd protein were made using the Bac-to-Bac system according to the manufacturer’s instructions (Invitrogen).

Rabbit polyclonal anti-human Dbf4, human Cdc7, human MCM2 and phospho-MCM2S139 antibodies were previously described (Tsuji et al., 2006). Rabbit polyclonal anti-XCdc7, XDbf4, XMCM10, XCdc45, XOrc2 and XDrf1 antibodies were kindly provided by Drs. Johannes Walter (Harvard Medical School, MA), Julian Blow (University of Dundee, UK) and Shou Waga (Osaka University, Japan), Ryuji Yamaguchi and John Newport (University of California, San Diego, CA) and Bill Dunphy (California Institute of Technology, CA). Anti-FLAG, Chk1, Histone H3B, Cdc2, phospho-Chk1S345, phospho-Histone H3BS10 and phospho-Cdc2Y15 antibodies were purchased from Sigma, Santa Cruz Biotechnology, Millipore and Cell Signaling Technology, respectively. All secondary antibodies used in the study were purchased from BD Biosciences.

Protein purification, immunoprecipitation, immunoblotting, and in vitro kinase assay

The human Cdc7/Dbf4, Cdc7/Drf1, Cdc7kd/Dbf4 and Cdc7kd/Drf1 kinase complexes were purified from insect Sf9 cells that were infected with the corresponding baculoviruses as previously described (Tsuji et al., 2006). GST-fused N-terminal region of MCM2 (MCM2M5: residues 1-178) and p27 were bacterially expressed and purified by a standard method (Tsuji et al., 2006). Total cell extraction (1% NP40), CSK extraction (CSK/0.5%TritonX100), immunoprecipitation, immunoblotting and kinase assay were performed as previously described (Jiang et al., 1999; Tsuji et al., 2006). Immunoblotting intensities were determined by Image-J imaging software (NIH).

Preparations of Xenopus egg extracts and demembraned sperm nuclei, immunodepletion, DNA replication assay and analysis of nuclear or chromatin-bound proteins

Xenopus interphase egg extracts and demembraned sperm nuclei were prepared as described (Lupardus et al., 2007). Immunodepletion, DNA replication assay and immunoblotting analysis of nuclear or chromatin bound proteins were performed as described (Lupardus et al., 2007). Band intensities from DNA replication assays were monitored by PhosphorImager (Molecular Dynamics).

Generation of tetracycline inducible HeLa cell lines overexpressing human Dbf4 and cell cycle analysis

HeLa-off cells stably expressing pTet-tTS (BD Biosciences) were transfected with pTRE2puro-FLAG-tagged Dbf4 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Puromycin-resistant clones were isolated and screened for their ability to express FLAG-Dbf4 in the presence or absence of tetracycline by immunoblotting analysis using anti-FLAG antibody. Of 17 clones analyzed, 2 clones (56α and 57α) expressed undetectable levels of FLAG-Dbf4 in the presence of tetracycline but high levels of FLAG-Dbf4 in the absence of tetracycline. FACS analysis was performed as described (Tsuji et al., 2006).

Supplementary Material

01

Acknowledgments

We thank Drs. Johannes Walter, Julian Blow, Shou Waga, Ryuji Yamaguchi, John Newport and Bill Dunphy for antibodies. T.T is very grateful to Drs. Johannes Walter, Ryuji Yamaguchi, Mark Mercola and Ann Foley for advice on the Xenopus system. E.L was supported by predoctoral training grant 2T32 CA77109-06A2. This work was supported by a grant from National Institutes of Health (GM67859) to W.J.

Footnotes

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