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. 2012 Jan 20;287(4):2531-43.
doi: 10.1074/jbc.M111.291104. Epub 2011 Nov 28.

Dbf4 is direct downstream target of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein to regulate intra-S-phase checkpoint

Alan Yueh-Luen Lee  1 , Takuya Chiba, Lan N Truong, An Ning Cheng, Johnny Do, Michael Jeffrey Cho, Longchuan Chen, Xiaohua Wu
Affiliations

Dbf4 is direct downstream target of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein to regulate intra-S-phase checkpoint

Alan Yueh-Luen Lee et al. J Biol Chem. .

Abstract

Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.

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Figures

FIGURE 1.
FIGURE 1.
Dbf4 is hyperphosphorylated in response to DNA damage in various cell lines. A, whole cell extracts were prepared from 293T cells and treated with or without protein phosphatase (pptase or mock) as described (39). Anti-Dbf4 Western blot analysis was performed. B, 293T, U2OS, T98G, and HeLa cells were treated without (No) or with IR (20 Gy, 30 min after), HU (1 mm, 24 h after), or UV light (30 J/m2, 1 h after). Whole cell extracts were prepared, and Western blot analysis for Dbf4 and Cdc7 was performed. C, whole cell extracts were prepared from 293T cells before (No) or after IR treatment (20 Gy, 30 min after), followed by treatment with or without protein phosphatase. Anti-Dbf4 Western blot analysis was performed.
FIGURE 2.
FIGURE 2.
DNA damage-induced Dbf4 hyperphosphorylation is mediated by ATM and ATR. A, 293T cells were transfected with empty vector (Vector or −), the ATM expression vector (ATM, left) or the ATR expression vector (+, right). Whole cell extracts were prepared after mock treatment (No) or exposure to IR (20 Gy, 30 min after) or HU (1 mm, 24 h after), and Western blotting for Dbf4 was performed. B, whole cell extracts were isolated from ATM-deficient cell line (FT169; atm−) or its derivative cell line (YZ5; ATM+) reconstituted with wild type ATM before (No) or after exposure to IR (20 Gy, 30 min after). Western blot analysis for Dbf4 was performed. C, fibroblast cell line GM847 and its derivative cell line carrying doxycycline-inducible ATR-KD (GM847-ATR-KD) were incubated with doxycycline (1 μg/ml) for 24 h and then treated with HU (1 mm, 24 h, +) or without (−) in the presence of doxycycline. Whole cell extracts were prepared, and Western blot analysis of Dbf4 was performed. D, schematic drawing of full-length Dbf4 containing eight ATM/ATR consensus phosphorylation sites, (S/T)Q sites, is represented. Four GST-fused Dbf4 fragments (SQ1–SQ4) containing one or two (S/T)Q sites are illustrated. E, ATM in vitro kinase assay was performed by using anti-FLAG immunoprecipitates from 293T cells that were transiently transfected with FLAG-tagged ATM. The immunoprecipitates were incubated with each of the glutathione-eluted purified GST-SQ fragments as indicated in the presence of [γ-32P]ATP. The autoradiogram shows the results of the in vitro kinase assay (left), and the Coomassie Blue-stained gel indicates the relative amount of protein added into the in vitro kinase reaction (right). F, 293T cells were transiently transfected with FLAG-tagged ATR, and the anti-FLAG immunoprecipitates were incubated with each of the glutathione-eluted purified GST-SQ fragment in the presence of [γ-32P]ATP. Phosphorylation of Dbf4 fragments and the input of the Dbf4 fragments are shown by autoradiogram (left) or Coomassie Blue-stained gel (right), respectively. G, in vitro ATM kinase assay was performed using purified GST-Dbf4 wild type fragments SQ1, SQ3, or SQ4, or the corresponding fragments with alanine substitution of serine or threonine residues at the (S/T)Q (SQ/TQ) sites as indicated. ATM-mediated phosphorylation of Dbf4 fragments was revealed by an 32P autoradiogram (top), and relative input for the GST-fused Dbf4 fragments is indicated by a Coomassie Blue-stained gel (bottom).
FIGURE 3.
FIGURE 3.
ATM and ATR phosphorylate Dbf4 at ATM/ATR consensus sites in vivo. A, U2OS cells were retrovirally infected with Myc-tagged wild type Dbf4 (left), FLAG-tagged wild type Dbf4 (WT), or the FLAG-Dbf4–3A (T449A, S502A, and S539A) and Dbf4-S539A mutants (right). Anti-Myc or anti-FLAG immunoprecipitation (IP) was performed before (No) or after IR treatment (20 Gy, 1 h after). Western blot analysis of anti-Myc immunoprecipitates (left) or anti-FLAG immunoprecipitates (right) was performed using affinity-purified polyclonal antibody recognizing phosphorylated Dbf4 at Ser-539 and monoclonal antibodies for Myc and FLAG tags. B, whole cell lysates were prepared from U2OS cells before (No) or after treatment with IR (20 Gy, 1 h after) or UV (50 J/m2, 1 h after). Western blot analysis using affinity-purified antibody recognizing phosphorylated Dbf4 at Ser-539 was performed. Western blotting against actin was used as a loading control. C, left, U2OS cells retrovirally infected with pMKO vector, ATR-shRNA, or ATM-shRNA were mock treated (No) or treated with IR (10 or 20 Gy, 1 h after) or UV light (50 J/m2, 1 h after). Whole cell lysates were analyzed by Western blot analysis using the phospho-specific antibody to Dbf4-Ser539. The expression of ATR and ATM after shRNA expression was revealed by Western blot analysis. Ku70 was used as a loading control. Right, U2OS cells expressing doxycycline-induced ATR-WT or ATR-KD were mock-treated (No) or treated with UV (50 J/m2, 1 h after) or HU (1 mm, 24 h). Whole cell extracts were prepared and immunoblotted using the indicated antibodies. D, U2OS stable cell lines were generated by retroviral infection to express FLAG-tagged wild type Dbf4 (WT) or the Dbf4 phosphomutants 3A (S449A/S502A/S539A), 5A (S226A/T265A/S449A/S502A/S539A), or S539A. Cell lysates were prepared before (No) and after treatment of IR (20 Gy, 1 h after) or UV (50 J/m2, 1 h after), and anti-FLAG Western blot analysis was performed.
FIGURE 4.
FIGURE 4.
ATM/ATR-directed Dbf4 phosphorylation is important for mediating the intra-S-phase checkpoint. U2OS cells were retrovirally infected with Myc-tagged wild type Dbf4, Dbf4-3A (T449A/S502A/S539A), or Dbf4-S539A, followed by inactivation of endogenous Dbf4 with one round of shRNA retroviral infection. These cells were treated with IR (10 Gy, 1 h after), and the rate of DNA synthesis was determined. Left, DNA synthesis rate is expressed as the percentages by normalizing to the wild type sample without IR treatment. The expression of Myc-tagged wild type Dbf4, Dbf4-3A, or Dbf4-S539A and the inactivation of endogenous Dbf4 by shRNAs are revealed by anti-Dbf4 Western blot analysis (top right). The cell cycle distribution of the cell lines expressing Myc-tagged wild type Dbf4, Dbf4-3A, or Dbf4-S539A with endogenous Dbf4 inactivated by shRNAs before IR is shown (bottom right). Error bars, S.D.
FIGURE 5.
FIGURE 5.
DNA damage does not affect MCM2 phosphorylation by Dbf4/Cdc7 in vivo and in vitro. A, immunoprecipitation of Dbf4 was performed before (No) or after treatment of 293T cells with IR (20 Gy, 1 h after), UV (50 J/m2, 1 h after), or HU (1 mm, 24 h), and immunoprecipitates (IP) were subjected to Western blot analysis using antibodies to Dbf4 and Cdc7. B, DDK in vitro kinase assay. 293T cells were transfected with FLAG-Dbf4/Cdc7WT or FLAG-Dbf4/Cdc7KD. 48 h after transfection, cells were treated with IR (20 Gy, 1 h after), HU (1 mm, 24 h), or UV light (50 J/m2, 1 h after) or without (No). FLAG-Dbf4/Cdc7WT or FLAG-Dbf4/Cdc7KD were immunoprecipitated by anti-FLAG antibody and used for the in vitro kinase assays. Autophosphorylation of Dbf4 was performed by incubating the anti-FLAG immunoprecipitates with [γ-32P]ATP (left), and the amount of FLAG-Dbf4 and Cdc7 in the anti-FLAG immunoprecipitates is revealed by Western blot analysis. Phosphorylation of MCM2 by DDK was carried out by using purified GST-MCM2 (amino acids 1–169) fragment or GST as substrate in the DDK in vitro kinase assay (right). Phosphorylation was revealed by autoradiography (32P), and the relative protein input is shown by Coomassie Blue staining (Input). C, left, T98G cells were synchronized by arresting in G0 after serum starvation (0.1% FBS for 48 h) and releasing into medium containing 10% FBS. Cells were harvested at the indicated time points after releasing, and the cell cycle profile is shown. Chromatin fractions at each time point were isolated and immunoblotted with an antibody to MCM2. Ku70 was used as a loading control. Asy, asynchronized cell population; MCM2un, unphosphorylated MCM2; MCM2p, phosphorylated MCM2. Right, U2OS cells were infected with adenoviruses encoding green fluorescent protein (Ad-GFP), p27 (Ad-p27), or Cdc7KD (Ad-Cdc7KD). The chromatin fractions were prepared 48 h after infection and analyzed by immunoblotting with anti-MCM2 antibody. Western blotting of Ku70 was used as a loading control. D, T98G cells were synchronized as described in C and released to the medium containing 10% FBS with or without drug. Cells without drug treatment were harvested at 0, 12, 16, or 20 h after releasing. Cells with drug treatment were harvested at 40 h after releasing into HU (1 mm) or aphidicolin (Aph; 1 μg/ml)-containing medium. The chromatin fractions were prepared, Western blot analysis was performed using the antibody recognizing MCM2, and Ku70 was used as a loading control. The cell cycle profile is indicated. E, 16 h after releasing from serum starvation, T98G cells were mock-treated (No) or treated with IR (20 Gy). The chromatin fractions were isolated from cells without treatment (No) or 1, 3, or 6 h after IR treatment. Western blot analysis was performed using an antibody recognizing MCM2, and Ku70 was used as a loading control.
FIGURE 6.
FIGURE 6.
The kinase activity of Dbf4/Cdc7 is important for protecting fork integrity. A, chromatin fractions were isolated from U2OS cells after mock treatment (No), or treatment with IR (20 Gy, 1 h after), HU (1 mm, 24 h after), thymidine (Thy; 2 mm, 24 h after), or aphidicolin (Aph; 1 μg/ml, 24 h after) and were subjected to Western blot analysis with antibodies to Dbf4 and Cdc7. Western blotting of Ku70 was used as a loading control. B, T98G cells were arrested in G0 by serum starvation (0.1% FBS, 48 h) and released into medium containing 10% FBS without drug or with HU (1 mm) or Aph (1 μg/ml) for 40 h as described in the legend to Fig. 4D. Chromatin fractions were isolated and immunoblotted with antibodies to Dbf4 and Cdc7. Ku70 was used as a loading control. Cell cycle profiles are shown in Fig. 5D. C, U2OS cells were mock-treated (No) or treated with HU (1 mm), and chromatin fractions were prepared at different time points (hours) after HU treatment. The samples were immunoblotted with anti-γH2AX and Ku70 antibodies. D, U2OS cells were retrovirally infected with pMKO vector or pMKO expressing shRNAs for Dbf4 or Cdc7. Subsequently, these cells were mock-treated (No) or treated with HU (1 mm, 8 h). Chromatin fractions were prepared and immunoblotted with anti-γH2AX and Ku70 antibodies (left). The expression of Dbf4 and Cdc7 with or without infection of shRNAs was shown by Western blot analysis of cell lysates, and actin was used as a loading control (right). E, U2OS cells were infected with adenovirus encoding green fluorescent protein (Ad-GFP) or Cdc7KD (Ad-Cdc7KD). 48 h after infection, cells were mock-treated (No) or treated with HU (1 mm, 12 h). Chromatin fractions were prepared and immunoblotted with antibodies to γH2AX, Cdc7, or Ku70. F, chromatin fractions were isolated from U2OS cells after mock treatment (No) or treatment with IR (20 Gy, 1 h after), UV light (50 J/m2, 1 h after), HU (1 mm, 24 h), thymidine (Thy; 2 mm, 24 h), or aphidicolin (Aph; 1 μg/ml, 24 h). Chromatin fractions were analyzed by Western blotting using antibodies recognizing Dbf4 and phosphorylated Dbf4 at Ser-539. Western blotting of Ku70 was used as a loading control. G, U2OS cells were retrovirally infected with FLAG-tagged wild type Dbf4 (WT), the Dbf4-3A mutant (3A), or the Dbf4-S539A mutant, followed by retroviral infection with shRNAs to endogenous Dbf4. Cells were mock-treated (No) or treated with HU (1 mm, 8 h), and chromatin fractions were isolated. Western blot analysis was performed using anti-γH2AX antibodies, and Ku70 was used as a loading control. H, U2OS cells expressing FLAG-Dbf4-WT, -3A, or -S539A alleles were mock-treated (No) or treated with HU (1 mm, 24 h). Chromatin fractions were isolated, and Western blot analysis was performed using anti-FLAG antibody. Ku70 was used as a loading control.
FIGURE 7.
FIGURE 7.
ATM/ATR-dependent Dbf4 phosphorylation is important for suppressing DNA rereplication. A, whole cell lysates were prepared from U2OS cells infected with Ad-GFP or Ad-Cdt1 using two different viral titers for 48 h (5 × 107 or 1 × 108 pfu/ml). The lysates were analyzed by Western blotting using the indicated antibodies with actin as a loading control. B, U2OS cells were infected with Ad-GFP or Ad-Cdt1 (5 × 107 pfu/ml), and whole cell lysates and chromatin fractions were prepared at different time points after infection. The prepared lysates and chromatin fractions were immunoblotted with the indicated antibodies, and Ku70 was used as a loading control. C, U2OS cells were retrovirally infected with vector pMKO (vec) or pMKO encoding shRNAs against ATR or ATM. These cells were subsequently infected with Ad-GFP or Ad-Cdt1 (5 × 107 pfu/ml), and whole cell lysates were prepared at 14 or 24 h after infection. Western blot analysis was performed using the antibodies recognizing phosphorylated Ser-539 of Dbf4 or Ku70 (top). The expression of ATR or ATM was examined by Western blot analysis (bottom). D, U2OS cells were infected with Ad-GFP or Ad-Dbf4–5A (S226A/T265A/T449A/S502A/S539A) using increased viral titers (5 × 107, 5 × 108, or 1 × 109 pfu/ml) for 48 h and subsequently treated with IR (20 Gy, 1 h after, +) or mock treatment (−). Western blot analysis was performed using antibodies specific for Dbf4-pS539 or FLAG tag. Ku70 was used as a loading control. E, U2OS cells were infected with Ad-GFP or Ad-Dbf4-5A (5 × 108 pfu/ml) for 24 h, followed by infection using Ad-GFP or Ad-Cdt1 (5 × 107 pfu/ml) for another 48 h. Infected cells were collected for FACS analysis. F, the expression of endogenous Dbf4 in U2OS cells expressing wild type Dbf4 or Dbf4-3A was suppressed by two rounds of shRNA retroviral infection, followed by adenoviral infection with Ad-GFP or Ad-Cdt1 (5 × 107 pfu/ml). FACS analysis was performed 48 h after adenoviral infection (left). Cdt1 expression was shown by anti-Cdt1 Western blot analysis with actin as a loading control (right).
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References

    1. Kelly T. J., Brown G. W. (2000) Regulation of chromosome replication. Annu. Rev. Biochem. 69, 829–880 - PubMed
    1. Bell S. P., Dutta A. (2002) DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 - PubMed
    1. Lei M., Tye B. K. (2001) Initiating DNA synthesis. From recruiting to activating the MCM complex. J. Cell Sci. 114, 1447–1454 - PubMed
    1. Labib K. (2010) How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells? Genes Dev. 24, 1208–1219 - PMC - PubMed
    1. Johnston L. H., Masai H., Sugino A. (2000) A Cdc7p-Dbf4p protein kinase activity is conserved from yeast to humans. Prog. Cell Cycle Res. 4, 61–69 - PubMed

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