DDB1 Targets Chk1 to the Cul4 E3 Ligase Complex in Normal Cycling Cells and in Cells Experiencing Replication Stress Free

Chk1 is a serine/threonine protein kinase that functions to maintain genome integrity in normal cycling cells and in cells exposed to replicative and genotoxic stress (1, 2). Chk1 regulates cell cycle progression, in part, by phosphorylating the Cdc25A protein phosphatase (1–4). Phosphorylation of Cdc25A by Chk1 targets it for ubiquitin-mediated proteolysis and prevents it from binding to and activating cyclin-dependent kinase 1/cyclin B1 inappropriately during the S and G₂ phases of the cell division cycle (1, 2, 5–7). Chk1 enforces cell cycle delays in cells experiencing replicative or genotoxic stress by inhibiting elongation of replication forks, by preventing firing of new origins of replication, and by blocking mitotic entry (8, 9). Loss of the Chk1/Cdc25A regulatory pathway disrupts both the DNA replication and DNA damage checkpoints (2). In addition to regulating DNA replication and mitotic entry, Chk1 has also been shown to stabilize stalled replication forks when DNA replication is impeded, to regulate transcription through phosphorylation of histone H3, and to regulate DNA repair by phosphorylating the Fanconi anemia protein FANCE (8, 10–12). Chk1 carries out its functions both in the nucleus and at the centrosome (13). Drugs that block Chk1 kinase activity or enhance its proteolysis by interfering with binding to heat shock protein 90 (HSP90) are currently being tested as anticancer agents (14–17).

Chk1 is regulated by reversible phosphorylation and by ubiquitin-mediated proteolysis. Under periods of replicative stress, the ATRIP/ATR module binds to single-stranded DNA and, together with Rad17 and the 9-1-1 complex, activates Chk1 in a Claspin-dependent manner (18–22). ATR directly phosphorylates Chk1 on two COOH-terminal residues: Ser³¹⁷ (S317) and Ser³⁴⁵ (S345; refs. 23, 24). The dephosphorylation of Chk1 is mediated by at least two phosphatases. These include PP2A, which maintains Chk1 in a hypophosphorylated state in normal cycling cells (25), and PPM1D (Wip1), which dephosphorylates Chk1 during checkpoint recovery (26). In addition, Cul1- and Cul4A-containing E3 ubiquitin ligases have been shown to ubiquitinate Chk1 during periods of prolonged replication stress (27). The Cullins assemble a large number of distinct ubiquitin ligases by binding to ROC1, a RING protein, and to several distinct targeting subunits that recruit substrates for ubiquitination (28). The targeting subunits that recruit Chk1 to Cul1 and Cul4A have not been identified. Here, we show that damaged DNA-binding protein 1 (DDB1), a triple β propeller adapter protein, targets the Cul4 E3 ubiquitin ligase complex to Chk1.

Cell lines and lysis buffer. HeLa and HEK293 cells were grown in DMEM (Invitrogen) supplemented with 10% bovine growth serum. Cells were lysed in MCLB [50 mmol/L Tris-HCl (pH 8.0), 100 mmol/L NaCl, 5 mmol/L EDTA, 0.5% NP40, and 2 mmol/L DTT] containing 1 mmol/L sodium orthovanadate, 10 mmol/L β-glycerophosphate, 1 μmol/L microcystin, 2 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, and 5 μg/mL leupeptin (25).

Chemicals and drugs. Cycloheximide, MG132, hydroxyurea, wortmannin, and caffeine were purchased from Sigma Chemical Co. Gö6976 and geldanamycin were purchased from Calbiochem. SN38 was purchased from Avachem.

Antibodies and Western blotting. Primary antibodies used in this study included mouse monoclonal anti-Chk1 (G-4, Santa Cruz Biotechnology), affinity-purified rabbit anti-Chk1 (24), mouse anti-β-catenin (BD Biosciences), goat anti-DDB1 (Abcam), rabbit anti-V5 (Abcam), rabbit anti-Cul4A (Rockland), rabbit anti-HA (Santa Cruz Biotechnology), mouse monoclonal anti-Flag (Sigma Chemical), mouse monoclonal anti-actin (Santa Cruz Biotechnology), mouse monoclonal anti–proliferating cell nuclear antigen (Santa Cruz Biotechnology), and rabbit anti-pS345 and anti-pS317 Chk1 (Cell Signaling). Flag M2 affinity beads were from Sigma Chemical. Secondary antibodies included anti-rabbit horseradish peroxidase (HRP; Zymed), anti-goat HRP (Zymed), and anti-mouse HRP (Jackson ImmunoResearch). Immunoblots were visualized by chemiluminescence using an enhanced chemiluminescence kit (GE Healthcare) according to the manufacturer's instructions and quantified by densitometry using ImageJ (29).

Plasmids. Chk1 [wild-type (WT)] was amplified by PCR as a XhoI fragment from pcDNA3-myc-Chk1 (24) and was cloned into the XhoI site of p3XFlag-CMV (Sigma Chemical) to generate pFlag₃Chk1 (WT). pFlag₃Chk1 (SA/SA), which encodes a mutant form of Chk1 containing alanines in place of S317 and S345, was generated by site-directed mutagenesis of pFlag₃Chk1 (WT) using the QuikChange Mutagenesis kit (Stratagene) as described (24). V5-tagged DDB1 and Cul4A expression plasmids were generated using the Gateway System (Invitrogen) and purchased cDNAs (Ultimate open reading frame clones; Invitrogen) to generate pcDNAnV5-DDB1 and pcDNAnV5-Cul4A. Plasmids encoding HA-ubiquitin (HA-Ub) have been described (30).

Isolation and identification of Chk1-associated proteins. HEK293 cells (1.2 × 10⁷) were transfected for 24 h with pFlag₃-CMV control plasmid or plasmid encoding Flag₃Chk1 (WT) using SuperFect (Qiagen) and incubated in the presence of 10 mmol/L hydroxyurea for 4 h. Lysates were prepared and incubated with Flag M2 affinity beads to generate control beads and Flag₃Chk1 beads. Hydroxyurea-treated lysates from HEK293 cells (4 × 10⁸) were prepared and incubated with control or Flag₃Chk1 beads overnight. Immunocomplexes were washed six times with MCLB, boiled, and resolved by SDS-PAGE. Gels were stained with colloidal Coomassie blue (Invitrogen) and selected bands were excised from the gel and analyzed by mass spectrometry as described (31). Briefly, after in-gel trypsinization, peptides were analyzed by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometry on a Finnigan LCQ DECA XP Plus quadrupole ion trap mass spectrometer at the Harvard Microchemistry Facility.

RNA interference experiments. Small interfering RNAs (siRNA) used in this study were purchased from Dharmacon and included a control siRNA (D00121002), or siRNAs specific for luciferase (D00140001), Cul4A (M01261000), Cul4B (M01796500), and DDB1 (M01289001). Approximately 2 × 10⁵ HeLa cells were seeded in each well of a six-well tissue culture dish. The following day, cells were transfected for 48 to 72 h with 100 nmol/L siRNAs using DharmaFECT 2 or DharmaFECT Duo reagents according to the manufacturer's instructions. For monitoring the ionizing radiation (IR)–induced G₂ checkpoint, asynchronously growing HeLa cells were transfected with siRNAs for 24 h and then incubated with DMSO or 200 nmol/L geldanamycin for another 24 h. Cells were then exposed to 10 Gy IR. In some cases, cells were also incubated with DMSO (vehicle) or Gö6976 (300 nmol/L) and harvested 9 h after IR.

Chk1 immunoprecipitations. Endogenous Chk1 was immunoprecipitated as described (24). For ectopic Flag₃Chk1 immunoprecipitations, 1× 10⁶ HeLa cells were seeded into 60-mm tissue culture dishes and the following day transfected with 2 μg pFlag₃Chk1, 4 μg pcDNAnV5-DDB1, and 1.5 μg pcDNAnV5-Cul4A using Lipofectamine 2000 (Invitrogen). Lysates were prepared 24 h later and incubated with protein A-agarose (Pierce) for 1 h. Precleared lysates were then incubated with Flag M2 affinity beads for 4 h at 4°C and precipitates were washed four times with MCLB. Flag₃Chk1 and associated proteins were eluted with 0.7 μg/μL of purified Flag₃ peptide (Sigma Chemical) in MCLB. Eluates were subjected to SDS-PAGE followed by Western blotting.

Chk1 ubiquitination in vitro. To generate Chk1 substrate, 1 × 10⁶ HeLa cells were transfected with 5 μg of pFlag₃Chk1 (WT) using Lipofectamine 2000. The following day, transfected cells were treated with 20 mmol/L hydroxyurea for 1 h to induce phosphorylation of Chk1. Cells were lysed in MCLB and lysates were precleared with protein A-agarose. Flag₃Chk1 was immunoprecipitated using 50 μL of anti-Flag beads at 4°C for 2 h. Immunoprecipitated Flag₃Chk1 was washed six times with MCLB, five times with LiCl buffer [0.5 mol/L LiCl, 50 mmol/L Tris (pH 8.0)], and two times with ubiquitination buffer [25 mmol/L Tris (pH 7.5), 5 mmol/L MgCl₂, 2 mmol/L NaF, 10 nmol/L okadaic acid, 2 mmol/L ATP, 0.6 mmol/L DTT]. Flag₃Chk1 was eluted in ubiquitination buffer containing 0.7 μg/μL of Flag₃ peptide. To immunoprecipitate Cul4A ligases, cells were treated with 20 mmol/L hydroxyurea for 1 h and lysed with MCLB and lysates were precleared with protein A-agarose. Cul4A was immunoprecipitated from 1 mg of total cell protein using 2 μL of anti-Cul4A antibody and 50 μL of protein A-agarose at 4°C for 4 h. Cul4A immunocomplexes were washed five times with MCLB and two times with ubiquitination buffer. Ubiquitin ligase reactions consisted of Cul4A immunocomplexes, Flag₃Chk1, and Sigma Chemical reagents: 12 μg bovine ubiquitin, 1.2 μg Flag ubiquitin, 60 ng E1, and 300 ng E2 (hUbc5c). Reactions were incubated at 37°C for 1 h and terminated by boiling the supernatant in loading buffer. Reactions were resolved by SDS-PAGE and analyzed by Western blotting.

Chk1 ubiquitination in vivo. HeLa cells (3 × 10⁵) were cotransfected for 48 h with plasmids encoding Flag₃Chk1 (0.5 μg), HA-Ub (1.5 μg), and 60 nmol/L of the indicated RNA interference (RNAi) using DharmaFECT Duo (Dharmacon). Transfected cells were then treated with 20 mmol/L hydroxyurea for 4 h. Cell lysates were prepared and incubated with protein A-agarose for 1 h. Precleared lysates were transferred to a fresh tube and incubated with Flag M2 affinity beads. Precipitates were washed, subjected to SDS-PAGE, and analyzed by Western blotting.

Cell synchrony. To synchronize HeLa cells at the G₁-S border, cells were treated with 2 mmol/L thymidine for 16 h. Cells were released from the block by washing twice with PBS followed by release into complete growth medium containing 24 μmol/L of both thymidine and 2′-deoxycytidine (Sigma Chemical). After 8 h, thymidine was added to the medium to a final concentration of 2 mmol/L, and cells were cultured for an additional 16 h. Cells were then rinsed twice with PBS and cultured in complete growth medium. A fraction of the cells was subjected to flow cytometry to determine cell cycle position. Cells were harvested by trypsinization and collected by centrifugation. Cells were washed once with PBS and fixed in 5 mL of 70% ethanol at 4°C. Cells were washed once with PBS/1% bovine serum albumin (BSA) and then incubated with 1 mL PBS/1% BSA containing 30 μg/mL propidium iodide and 0.25 mg/mL RNase A for 1 h at room temperature. Cells were analyzed for DNA content by flow cytometry using a FACSCalibur (BD Biosciences). The data were analyzed using CellQuest Analysis software (BD Biosciences).

DDB1 identified as a novel Chk1-interacting protein. A proteomic approach was used to identify novel upstream regulators and/or downstream targets of human Chk1. Flag-tagged Chk1 was affinity purified from hydroxyurea-treated HEK293 cells and then incubated in lysates prepared from hydroxyurea-treated HEK293 cells. As a control, cells transfected with the pFlag₃ vector were taken through the same procedure. As seen in Fig. 1A, a protein of ∼130 kDa selectively copurified with Flag-tagged Chk1 (lane 2) but not control beads (lane 1). Mass spectrometry of this protein identified 26 distinct peptides from the human DDB1 protein (Supplementary Fig. S1). These peptides corresponded to sequences spanning the entire DBB1 protein sequence.

DDB1 is part of an E3 ligase complex that includes the cullin proteins Cul4A and Cul4B (28, 32). Interestingly, Cul4A has been reported to target Chk1 for ubiquitin-mediated proteolysis during a DNA damage checkpoint response (27). However, the subunits that target Cul4A to Chk1 have not been identified. Therefore, we tested whether interactions between Chk1 and Cul4A/DDB1 complexes could be detected in vivo. Endogenous Chk1 was immunoprecipitated from vehicle- or hydroxyurea-treated HeLa cells and precipitates were analyzed for the presence of DDB1 and Cul4A by Western blotting. As seen in Fig. 1B, both DDB1 and Cul4A coprecipitated with Chk1 (lane 4) and associations were enhanced in hydroxyurea-treated cells (lane 5). Similar observations were made when Chk1 was coproduced with Cul4A and DDB1 in vivo (Fig. 1C). Interestingly, enhanced associations were detected when Chk1 was coproduced with both Cul4A and DDB1 (lane 10) compared with either subunit alone (lanes 11 and 12) and hydroxyurea treatment significantly enhanced interactions between Chk1 and Cul4A/DDB1 (lanes 14–16). On hydroxyurea removal, a decrease in the interactions between Chk1 and Cul4A/DDB1 was observed coincident with a decrease in Chk1 phosphorylation on S317 (Supplementary Fig. S2A).

Chk1 interactions with Cul4A-DDB1 are regulated by phosphorylation. In cells experiencing replicative stress, the ATR protein kinase phosphorylates Chk1 on S317 and S345 (23, 24, 33–35). This pathway can be blocked by expressing a mutant of Chk1 containing alanine in place of both of these serine residues (SA/SA) or by treating cells with wortmannin or caffeine, inhibitors of the phosphatidylinositol 3-kinase–related family, which include ATR and ATM (24, 36–38). As seen in Fig. 2A, interactions between Chk1 and Cul4A/DDB1 were impaired but not completely eliminated by mutating both S317 and S345 (compare lanes 2 and 3). Wortmannin reduced the binding of Cul4A/DDB1 to both WT Chk1 and the SA/SA mutant (compare lanes 2–5 and 3–6), as did caffeine (compare lanes 2–8 and 3–9). Total levels of Chk1 remained constant and phosphorylation of Chk1 on S317 was reduced under these experimental conditions (Supplementary Fig. S2B). Treatment of cells with Gö6976, a Chk1 inhibitor that enhances Chk1 phosphorylation on S317 and S345 in vivo (25), increased binding of the Cul4A/DDB1 ligase to Chk1 WT but not to Chk1 SA/SA (Fig. 2A , compare lanes 2–11 and lanes 3–12). Enhanced binding between endogenous Chk1 and endogenous Cul4A/DDB1 was also observed in response to Gö6976 treatment (Supplementary Fig. S2C). These results show that phosphorylation of Chk1 on S317 and S345 contributes to, but is not essential for, its interactions with Cul4A/DBB1.

Next, experiments were performed to monitor the association between Chk1 and the Cul4A/DDB1 complex as a function of the cell division cycle. Cells were arrested at the G₁-S border by a double thymidine block and release protocol. The association between Chk1 and Cul4A/DDB1 was monitored by immunoprecipitating endogenous Chk1 and testing for the coprecipitation of DDB1 and Cul4A by immunoblotting. As seen in Supplementary Fig. S3A, enhanced interactions were detected during the S and G₂ phases of the cell division cycle.

We recently reported that Chk1 is continually phosphorylated on S317 and S345 by ATR during the S and G₂ phases of the cell division cycle (25). However, Chk1 phosphorylation on these residues is not readily observed because PP2A continuously dephosphorylates Chk1 on S317 and S345. Furthermore, in cells knocked down for PP2A, Chk1 phosphorylation increases but Chk1 protein levels decrease (25). We asked if inhibition of the proteosome could restore Chk1 levels in PP2A-deficient cells. MG132 treatment was observed to partially rescue Chk1 levels in PP2A-deficient cells (Supplementary Fig. S3B and C). We next asked whether Cul4A/DDB1 was responsible for targeting Chk1 to the proteosome in PP2A-deficient cells (Fig. 2B). As expected, loss of PP2A resulted in enhanced S345 phosphorylation and a concomitant 50% decrease in Chk1 levels (compare lanes 1 and 2). In contrast, Chk1 levels rose in Cul4A/DDB1–deficient cells (compare lanes 1 and 4). Importantly, Chk1 levels were partially restored when PP2A-deficient cells were knocked down for the Cul4A/DDB1 E3 ligase (compare lanes 2 and 3). These results support the conclusion that Chk1 phosphorylation promotes its interactions with Cul4A/DDB1 and that Cul4A/DDB1 targets Chk1 for ubiquitination and proteosomal degradation.

Cul4A and DDB1 are required for Chk1 ubiquitination in vitro and in vivo. Cul4A is a member of the cullin family of ubiquitin-protein ligases and DDB1 is one of its targeting subunits. We assessed whether Chk1 could be directly ubiquitinated by the Cul4A/DDB1 ligase by performing ubiquitination reactions in vitro (Fig. 3A and B). Purified Flag₃Chk1 was used as substrate and RNAi-treated cell lysates were used as a source of Cul4A complexes. Ubiquitination of Chk1 was not observed when Chk1 precipitates were incubated in the presence of Cul4A precipitates but in the absence of purified E1 and E2 (Fig. 3B , lane 2). A low level of Chk1 ubiquitination was observed when Chk1 precipitates were incubated with purified E1 and E2, indicating that Chk1 precipitates also contain E3 ligase activity (lane 6). Chk1 ubiquitination was enhanced in complete reactions containing Cul4A, E1, and E2 (lane 3) and was diminished when Cul4A was precipitated from cells knocked down for either Cul4A or DDB1 (lanes 4 and 5).

Next, we tested the requirement for Cul4A and DBB1 for Chk1 ubiquitination in vivo. Cells expressing Flag₃Chk1 and HA-Ub were transfected with siRNAs and Chk1 precipitates were analyzed for the presence of HA-Ub. Chk1 ubiquitination was diminished in cells knocked down for either DDB1 (Fig. 3D) or both DDB1 and Cul4A (Supplementary Fig. S4A). Taken together, these results show that Cul4A/DDB1 ubiquitinates Chk1 in vitro and that efficient Chk1 ubiquitination in vivo requires Cul4A and DDB1.

Knockdown of Cul4 or DDB1 stabilizes Chk1 in vivo. Next, experiments were performed to test whether the half-life of Chk1 is regulated by the Cul4A/DDB1 E3 ligase in vivo. To this end, HeLa cells were treated with control siRNAs or siRNAs specific for either Cul4 or DDB1 and the half-life of Chk1 was measured in the presence of cycloheximide (Fig. 4). These experiments were performed in both normal cycling cells (Fig. 4A and B) and hydroxyurea-treated cells (Fig. 4C and D). The half-life of Chk1 was determined to be ∼6 hours in normal cycling HeLa cells (Fig. 4B) and ∼5 hours in hydroxyurea-treated cells (Fig. 4D). Loss of either Cul4 or DDB1 extended the half-life of Chk1 to >10 hours in normal cycling cells (Fig. 4B) and to >8 hours in hydroxyurea-treated cells (Fig. 4D). Additionally, the half-life of Chk1 was extended when DDB1 and/or Cul4A were knocked down in hydroxyurea-treated A549, HEK293, and HCT116 cells (data not shown).

Chk1 turnover induced by replication stress or HSP90 inhibition is dependent on Cul4A/DDB1 E3 ligase. Camptothecin, a topoisomerase I inhibitor, induces Chk1 degradation in vivo (27). We monitored the effects of SN38, the active metabolite of camptothecin, on Chk1 levels in control cells and in cells deficient for the Cul4A/DDB1 ligase (Fig. 5A). Importantly, SN38 treatment was less effective at inducing Chk1 loss in cells knocked down for Cul4 and DDB1. The half-life of Chk1 was ∼4 hours in control cells treated with SN38 and cycloheximide (Supplementary Fig. S4B). In contrast, the half-life of Chk1 was extended to >8 hours in DDB1-deficient cells treated with SN38 and cycloheximide (Supplementary Fig. S4B).

Treatment of cells with geldanamycin, a HSP90 inhibitor, also causes Chk1 turnover. HSP90 binds Chk1 (39) and inhibition of Chk1/HSP90 interactions leads to Chk1 proteolysis via the proteosome (Supplementary Fig. S4C; ref. 40). We tested whether knockdown of Cul4A/DDB1 impaired Chk1 proteolysis in geldanamycin-treated cells (Fig. 5B). As expected, knockdown of Cul4A/DDB1 resulted in Chk1 stabilization (compare lanes 1 and 4) and geldanamycin treatment reduced Chk1 levels to ∼17% that observed in control RNAi-treated cells (compare lanes 1 and 2). Importantly, the ability of geldanamycin to induce Chk1 proteolysis was impaired in Cul4A/DDB1–deficient cells (compare lanes 2 and 3).

The Chk1/Cul4A/DDB1 pathway regulates the IR-induced G₂ checkpoint. Lastly, we investigated the contribution made by the Chk1/Cul4A/DDB1 regulatory pathway to the IR-induced G₂ checkpoint. Knockdown of Cul4A/DDB1 was used to maintain Chk1 levels in the presence of the HSP90 inhibitor geldanamycin. Effects on the IR-induced G₂ checkpoint were then evaluated in control cells (low Chk1 levels) and in cells deficient in Cul4A/DDB1 (higher Chk1 levels). The dependency of the checkpoint response on Chk1 was determined by carrying out the experiments in the presence and absence of the Chk1 inhibitor Gö6976. Levels of phosphohistone H3, a mitosis-specific marker, were monitored to assess the integrity of the checkpoint in these experiments (Fig. 5C).

Cells treated with siRNAs specific for Cul4 and DDB1 (to interfere with Chk1 turnover) or with control siRNAs were incubated with the HSP90 inhibitor to induce Chk1 turnover. As expected, Chk1 levels were lower in geldanamycin-treated control cells (lane 2) compared with geldanamycin-treated Cul4A/DDB1–deficient cells (lane 6), supporting a role for Cul4A/DDB1 in regulating Chk1 turnover. These cells were then exposed to IR to activate the IR-induced G₂ checkpoint. Because Chk1 is an essential component of this checkpoint, we predicted that geldanamycin-treated control cells containing less Chk1 would be impaired in their ability to arrest in G₂ following exposure to IR. Indeed, phosphohistone H3 levels were higher in control cells (lane 2) relative to Cul4A/DDB1–deficient cells (lane 6). To determine if the difference was due to Chk1, the experiment was repeated in the presence and absence of the Chk1 inhibitor Gö6976. Inhibition of Chk1 in control cells did not significantly affect levels of phosphohistone H3, presumably due to the already low levels of Chk1 in these cells (compare Chk1 levels in lanes 2 and 3). In contrast, inhibition of Chk1 in Cul4A/DDB1–deficient cells induced checkpoint bypass as indicated by the higher levels of phosphohistone H3 (compare lanes 6 and 7). These experiments confirm that Chk1 levels can be modulated in vivo by perturbing Cul4A/DDB1 and show that perturbation of the Chk1/Cul4A/DDB1 regulatory pathway affects the ability of cells to respond appropriately to DNA double-strand breaks.

The preservation of genome integrity within individual cells is essential for organismal homeostasis. Cells activate signaling pathways known as checkpoints to arrest cell cycle progression when their genome integrity is compromised. This can occur during a normal division cycle when, for example, a replication fork temporarily stalls or in response to a direct assault on the genome resulting in either single- or double-strand breaks. At some point, these signaling pathways must be turned off in order for DNA replication and cell division to resume. As part of the recovery process, Claspin, a Chk1 regulator, is ubiquitinated and degraded (41); Chk1 is inactivated by dephosphorylation of S317 and S345 (26), or alternatively, activated Chk1 is degraded in a proteosome-dependent manner (27).

Here, we show that the Cul4A/DDB1 complex ubiquitinates Chk1 and regulates Chk1 abundance in normal cycling cells and when cells are exposed to exogenous assaults that induce replication stress. Affinity purification of Flag-tagged Chk1 coupled with mass spectrometry identified DDB1 as a Chk1-associated protein. Interactions between endogenous Chk1 and endogenous Cul4A/DDB1 were shown to occur in normal cycling cells and to be enhanced by replication stress. In addition, the half-life of Chk1 was extended in cells knocked down for either DDB1 or Cul4A. The Cul4A/DDB1 complex is proposed to target substrates by direct interaction with DDB1 (42) or through a family of adapter proteins called DDB1- and Cul4-associated factors (DCAFs; refs. 32, 43, 44). Although we showed that Chk1 phosphorylation is important for its interaction with Cul4A/DDB1, we do not yet know whether the interactions between Chk1 and DDB1 are direct or are mediated by a DCAF. To date, our attempts to reconstitute interactions between Chk1 and DDB1 with purified proteins in vitro have failed. Interestingly, two novel WD40-containing proteins were identified in our Chk1 proteomic screen. WD40 domains are a common feature of DCAFs (45), and future experiments will be directed at determining whether either of these proteins mediates interactions between Chk1 and DDB1.

The identification of Chk1 as a Cul4A/DDB1 target adds to a growing list of cellular substrates targeted to Cul4 by DDB1, including the nucleotide excision repair proteins DDB2 and XP group C (46, 47), the DNA replication licensing factor Cdt1 (42, 48, 49), the cell cycle regulator p27 (50), the tumor suppressor proteins TSC2 (51) and Merlin (52), the transcription factors c-Jun and signal transducer and activator of transcription proteins (53, 54), as well as a subset of histones (55, 56). Thus, the Cul4-DDB1-Roc1 complex regulates several critical cellular processes, including nucleotide excision repair, transcription, cell cycle progression, checkpoints, and DNA replication. The importance of the Cul4A/DDB1 E3 ligase to the cell division cycle is underscored by the fact that several pathogenic viruses subvert the Cul4A/DDB1 complex to benefit their own replicative life cycles and DDB1 expression has been reported to be significantly decreased in adrenocortical carcinomas (57).

Chk1 enforces S-phase delays in cells experiencing replicative or genotoxic stress by inhibiting elongation of replication forks and by preventing firing of new origins. Thus, Cul4A/DDB1 is predicted to contribute to the checkpoint recovery process by facilitating the ubiquitin-mediated proteolysis of Chk1, thereby enabling faithful resumption of DNA replication and cell cycle progression following recovery from replicative stress and DNA damage.

No potential conflicts of interest were disclosed.

Grant support: NIH GM047017. H. Piwnica-Worms is an Investigator of the Howard Hughes Medical Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Janis L. Watkins and Christine E. Ryan for preparing plasmid stocks and manuscript editing.