Journal: International Journal of Biological Sciences

Article Title: SIRT1 Deacetylates TopBP1 and Modulates Intra-S-Phase Checkpoint and DNA Replication Origin Firing

doi: 10.7150/ijbs.11066

Figure Lengend Snippet: SIRT1 interacting proteins.

Article Snippet: Western blotting was carried out with a Licor (Lincoln, Nebraska) or using the conventional ECL system with antibodies against CHK1 (Cell signaling), CDC7 (Cell Signaling), SIRT1 (Millipore), TopBP1 (Bethyl Lab), PARP (Santa Cruz), Flag antibody (Sigma, M2) and acetyl-lysine (Millipore and Cell Signaling).

Techniques:

Journal: International Journal of Biological Sciences

Article Title: SIRT1 Deacetylates TopBP1 and Modulates Intra-S-Phase Checkpoint and DNA Replication Origin Firing

doi: 10.7150/ijbs.11066

Figure Lengend Snippet: SIRT1 interacts with proteins involved in the regulation of DNA replication. (A, B) Interaction between ectopically over-expressed Flag-SIRT1 with TopBP1 (A) and CDC7 (B) as detected by immuno-precipitation. (C-E) Reciprocal immuno-precipitation with endogenous proteins to confirm interaction between SIRT1 versus TopBP1,CHK1 and PARP1 (C); TopBP1 versus SIRT1 (D); and CHK1 versus SIRT1 (E). (F) DNA replication stress enhances interaction of SIRT1 with TopBP1 and CHK1. 293T cells were treated with different doses of HU for 4 hours. Then SIRT1 was immuno-precipitated and TopBP1 and CHK1 were detected by western blot.

Article Snippet: Western blotting was carried out with a Licor (Lincoln, Nebraska) or using the conventional ECL system with antibodies against CHK1 (Cell signaling), CDC7 (Cell Signaling), SIRT1 (Millipore), TopBP1 (Bethyl Lab), PARP (Santa Cruz), Flag antibody (Sigma, M2) and acetyl-lysine (Millipore and Cell Signaling).

Techniques: Immunoprecipitation, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint

doi: 10.1007/s00018-022-04374-3

Figure Lengend Snippet: Down-regulation of CK2α affects the expression of cell cycle regulatory proteins implicated in the response to DNA replication stress. A Scheme summarizing the eukaryotic replisome complex coordinating DNA replication. DNA replication is carried out by specific DNA polymerases (Pol) on the leading and legging strands, respectively. Stretches of ssDNA are coated and stabilized by replication protein A (RPA). Unwinding of the DNA strands is carried out by minichromosome maintenance protein complex (MCM2-7) associated with cell division cycle 45 (CDC45) and go-ichi-ni-san (GINS) proteins. Altogether, they form the CMG complex. DNA replication is a process carried out with high fidelity. In order to accomplish this, eukaryotic cells express checkpoint proteins whose task is to closely monitor the genome and detect errors or problems during DNA replication. Checkpoint proteins are evolutionary well conserved and include ATR, ATRIP, CHK1, CLSPN, CDK2, Cdc7 which, altogether, mainly respond to stretches of ssDNA. Rad9–Hus1–Rad1 (9–1–1) is a heterotrimeric clamp on DNA sufficient to facilitate the interaction between ATR, its partner ATRIP, TOBP1 and CLSPN. During activation of a stress response following perturbation of DNA replication, CLSPN docks with CHK1 the major checkpoint effector kinase, contributing to promote the activation of surveillance mechanisms at the replisome. B , C Cells were incubated with vehicle or 1 μg/ml Dox for 72 h and then, where indicated, added 3 mM HU for 4 h ( B ), 6 h or 24 h ( C ). Harvested cells were processed as described in the materials and methods and whole cell lysates were analysed probing Western blot membranes with antibodies directed against proteins indicated in the figure. Experiments were performed at least three times obtaining similar results. β-Actin detection was used as loading control. One representative experiment is shown. Exp: exposure. Where indicated, the intensity of protein bands was quantified by densitometric analysis using ImageJ software (NIH) and calculated in percentage by assigning a value of 100% to specific bands as indicated in the figure

Article Snippet: Proteins were detected by incubating PVDF membranes with the following primary antibodies: rabbit polyclonal anti-phospho-NF-κB S529 (ab47395, Abcam, Cambridge, United Kingdom), rabbit monoclonal anti-phospho-CHK1 S345 (2348, Cell Signaling Technology), mouse monoclonal anti-CHK1 (2360, Cell Signaling Technology), rabbit monoclonal anti-cyclin E1 (20808, Cell Signaling Technology), mouse monoclonal anti-p53 (2524, Cell Signaling Technology), rabbit polyclonal anti-phospho-p53 S15 (9284, Cell Signaling Technology), rabbit polyclonal anti-Cdc7 (3603, Cell Signaling Technology), rabbit monoclonal anti-NF-κB (8242, Cell Signaling Technology), rabbit monoclonal anti-CDC45 (11881, Cell Signaling Technology), rabbit monoclonal anti-phospho-histone H2A.X S139 (9718, Cell Signaling Technology), mouse monoclonal anti-α-Tubulin (3873, Cell Signaling Technology), rabbit monoclonal anti-histone H3 (4499, Cell Signaling Technology); rabbit polyclonal anti-cyclin A (sc-751, Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal anti-ATR (sc-515173, Santa Cruz Biotechnology), mouse monoclonal anti-ATRIP (sc-365383, Santa Cruz Biotechnology), mouse monoclonal anti-MCM7 (sc-9966, Santa Cruz Biotechnology), mouse monoclonal anti-CLSPN (sc-376773, Santa Cruz Biotechnology), goat polyclonal anti-MCM3, mouse monoclonal anti-CDK2 (sc-6248, Santa Cruz Biotechnology), mouse monoclonal anti-HDAC2 (sc-9959, Santa Cruz Biotechnology), and mouse monoclonal anti-β-actin (A-5441, Sigma-Aldrich).

Techniques: Expressing, Activation Assay, Incubation, Western Blot, Software

Journal: Cellular and Molecular Life Sciences

Article Title: Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint

doi: 10.1007/s00018-022-04374-3

Figure Lengend Snippet: CK2α is required for DNA replication stress response. A H9c2-CK2α-44 cells were treated with vehicle or 3 mM HU for the indicated times. Whole cell lysates were subjected to immunoprecipitation (IP) with IgG (negative control experiment) or rabbit anti-CDC45 antibody. Samples were analysed by Western blot employing antibodies recognizing the proteins indicated in the figure. Whole cell lysate was also included in the analysis (Input) corresponding to 1% of the extract (control cells) employed in the immunoprecipitations. B FACS analysis of cells after synchronization by serum starvation (0.1% foetal bovine serum in the growth medium) for 48 h. Cells were released from cell cycle arrest when cultured again in the presence of complete growth medium. They were subsequently harvested at the indicated time-points and analysed. * P < 0.05 with respect to control cells at 18 h, and 20 h, respectively. C Cells were treated as indicated in B and in the presence or absence of Dox to induce down-regulation of CK2α. Whole cell lysates were subjected to immunoprecipitation with anti-CDC45 antibody. Samples were subsequently analysed as described in A . D–F Cells were left untreated or incubated with Dox for 72 h prior adding 3 mM HU for 4 and 24 h, respectively. Immunoprecipitation and Western blot analysis were then carried out as in A employing antibodies against the indicated proteins. G Cells were grown in the presence or absence of Dox for 72 h. After 24 h from the addition of Dox, cells were transfected with FLAG-CLSPN and HA-CHK1. 4 h before harvesting, cells were added HU as indicated in the figure. Immunoprecipitation and Western blot analysis were then carried out as described in A . H Whole cell lysates from H9c2-CK2α-44 cells were subjected to immunoprecipitation employing rabbit polyclonal anti-CK2α serum (results on the left side of the figure) or goat anti-CK2α antibody (results on the right side of the figure). Western blot analysis was subsequently carried out as in A . * Denotes non-specific band signal. All experiments were repeated at least three times obtaining similar results. Representative experiments are shown. Where indicated, the intensity of the protein bands was quantified by densitometric analysis as described in Fig. . Because of non-specific background signal in some results, the densitometric analysis of protein bands from the control experiments (IgG) is shown

Article Snippet: Proteins were detected by incubating PVDF membranes with the following primary antibodies: rabbit polyclonal anti-phospho-NF-κB S529 (ab47395, Abcam, Cambridge, United Kingdom), rabbit monoclonal anti-phospho-CHK1 S345 (2348, Cell Signaling Technology), mouse monoclonal anti-CHK1 (2360, Cell Signaling Technology), rabbit monoclonal anti-cyclin E1 (20808, Cell Signaling Technology), mouse monoclonal anti-p53 (2524, Cell Signaling Technology), rabbit polyclonal anti-phospho-p53 S15 (9284, Cell Signaling Technology), rabbit polyclonal anti-Cdc7 (3603, Cell Signaling Technology), rabbit monoclonal anti-NF-κB (8242, Cell Signaling Technology), rabbit monoclonal anti-CDC45 (11881, Cell Signaling Technology), rabbit monoclonal anti-phospho-histone H2A.X S139 (9718, Cell Signaling Technology), mouse monoclonal anti-α-Tubulin (3873, Cell Signaling Technology), rabbit monoclonal anti-histone H3 (4499, Cell Signaling Technology); rabbit polyclonal anti-cyclin A (sc-751, Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal anti-ATR (sc-515173, Santa Cruz Biotechnology), mouse monoclonal anti-ATRIP (sc-365383, Santa Cruz Biotechnology), mouse monoclonal anti-MCM7 (sc-9966, Santa Cruz Biotechnology), mouse monoclonal anti-CLSPN (sc-376773, Santa Cruz Biotechnology), goat polyclonal anti-MCM3, mouse monoclonal anti-CDK2 (sc-6248, Santa Cruz Biotechnology), mouse monoclonal anti-HDAC2 (sc-9959, Santa Cruz Biotechnology), and mouse monoclonal anti-β-actin (A-5441, Sigma-Aldrich).

Techniques: Immunoprecipitation, Negative Control, Western Blot, Cell Culture, Incubation, Transfection

Journal: Cellular and Molecular Life Sciences

Article Title: Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint

doi: 10.1007/s00018-022-04374-3

Figure Lengend Snippet: Model of CK2α-mediated regulation of cell cycle checkpoint response following induction of DNA replication stress. A Multiple replication factors contribute to regulate the DNA replication stress response preventing DNA damage and allowing completion of DNA replication. The slowing down or stalling of replication forks exposes ssDNA stretches which attract RPA whose function is to coat and stabilize these segments of DNA. It follows the recruitment of ATR–ATRIP complex which, in turn, phosphorylates CHK1 activating the checkpoint signalling. CLSPN interacts with CHK1 contributing to efficient ATR-mediated phosphorylation and activation of CHK1. We provide evidence showing that CK2α interacts with both CLSPN and CHK1 and stabilizes their association. The replisome is connected to DNA replication stress checkpoint machinery. Evidence shows that several protein kinases including Cdc7, CDK2 and ATR, phosphorylate various components of the MCM protein complex. In particular, ATM/ATR-dependent MCM phosphorylation primarily responds to replication stress. Besides CLSPN, efficient phosphorylation of CHK1 is mediated by other proteins including MCM7, RAD17, TopBP1 and the MRN complex. We found that CK2α interacts with MCM7 and CDC45 contributing to their assembly and localization on chromatin during normal cell cycle and upon induction of DNA replication stress, respectively. B We propose that CK2α plays a key role in maintaining the integrity of the replisome by stabilizing the association of specific replication factors and ensuring optimal activation of the ATR-CHK1 checkpoint pathway upon induction of replication stress. Down-regulation of CK2α leads to destabilization of the aforementioned complex formations which results in compromised DNA checkpoint response

Article Snippet: Proteins were detected by incubating PVDF membranes with the following primary antibodies: rabbit polyclonal anti-phospho-NF-κB S529 (ab47395, Abcam, Cambridge, United Kingdom), rabbit monoclonal anti-phospho-CHK1 S345 (2348, Cell Signaling Technology), mouse monoclonal anti-CHK1 (2360, Cell Signaling Technology), rabbit monoclonal anti-cyclin E1 (20808, Cell Signaling Technology), mouse monoclonal anti-p53 (2524, Cell Signaling Technology), rabbit polyclonal anti-phospho-p53 S15 (9284, Cell Signaling Technology), rabbit polyclonal anti-Cdc7 (3603, Cell Signaling Technology), rabbit monoclonal anti-NF-κB (8242, Cell Signaling Technology), rabbit monoclonal anti-CDC45 (11881, Cell Signaling Technology), rabbit monoclonal anti-phospho-histone H2A.X S139 (9718, Cell Signaling Technology), mouse monoclonal anti-α-Tubulin (3873, Cell Signaling Technology), rabbit monoclonal anti-histone H3 (4499, Cell Signaling Technology); rabbit polyclonal anti-cyclin A (sc-751, Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal anti-ATR (sc-515173, Santa Cruz Biotechnology), mouse monoclonal anti-ATRIP (sc-365383, Santa Cruz Biotechnology), mouse monoclonal anti-MCM7 (sc-9966, Santa Cruz Biotechnology), mouse monoclonal anti-CLSPN (sc-376773, Santa Cruz Biotechnology), goat polyclonal anti-MCM3, mouse monoclonal anti-CDK2 (sc-6248, Santa Cruz Biotechnology), mouse monoclonal anti-HDAC2 (sc-9959, Santa Cruz Biotechnology), and mouse monoclonal anti-β-actin (A-5441, Sigma-Aldrich).

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells

doi: 10.1038/ncomms12135

Figure Lengend Snippet: ( a ) Schematic diagram of various truncated or mutant derivatives of Claspin generated. BP1 and BP2: basic patch1 and 2. CKBD: Chk1 binding domain. ( b ) Mutant Claspin polypeptides tagged with 3x Flag at the C termini were expressed in 293T cells and pulled down with M2 Flag beads (Flag IP). Co-pulled down proteins were analysed by western blotting to detect the proteins indicated. Asterisk, IgG; black arrowheads, full-length or mutant Claspin polypeptides pulled down by Flag antibody. ( c ) Loss of interaction with Cdc7 in Cdel1 and Cdel6 mutants, whose structures are shown in the upper drawing. Interaction was examined as in b .

Article Snippet: Anti-PCNA (sc-56, 1:200), anti-Pol α (sc-5920, 1:200), anti-LaminB (sc-6216, 1:200), anti-ATR (sc-1887 1:200), anti-Chk1 (sc-8408, 1:200) and anti-MCM2 (sc-9839, 1:200) antibodies were from Santa Cruz Biotechnology; anti-TopBP1 (A300-111A, 1:1,000) and anti-MCM2 S53 (A300-756A, 1:1,000) antibodies were from Bethyl; anti-Cdc7 (K0070-3, 1:1,000) and anti-Flag (M185-3L, 1:1,000) antibodies were from MBL; anti-Chk1 S345 (#2341, 1:1,000), anti-tubulin (T5168, 1:1,000), anti-myc (04362-34, 1:1,000) and anti-HA (16B12, 1:1,000) antibodies were from Cell Signaling, Sigma-Aldrich, Nacalai Tesque and Abcam, respectively.

Techniques: Mutagenesis, Generated, Binding Assay, Western Blot

Journal: Nature Communications

Article Title: Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells

doi: 10.1038/ncomms12135

Figure Lengend Snippet: ( a ) Amino-acid sequences of aa988–1,086 (acidic patch, grey box) of Claspin. Acidic amino acids are underlined. In DE/A mutant, all these residues were replaced with alanine. ( b ) Wild-type (WT) and mutant Claspin indicated were expressed in 293T cells and were pulled down with M2 Flag beads. Associated proteins were analysed by western blotting to detect the proteins indicated. Values under each panel in lanes 6, 7 and 8 are the amount of each coimmunoprecipitated protein in Flag IP relative to that of the input protein. The values for the wild-type Claspin are taken as 1.00. ( c ) Purified wild-type or DE/A mutant Claspin was mixed with purified Cdc7-ASK complex and pulled down by Dynabeads conjugated with anti-Flag antibody. Immunoprecipated materials were analysed by western blotting, as shown. ( d ) Stable clones of f/− MEF cells expressing WT, DE/A and PIP were infected with Ad-Cre (+) or non-treated (−) for 2 days and 1.0 × 10 5 cells were passaged to new plates. The cells were then harvested at 2 days (Day 4 from Ad-Cre infection) and 3 days (Day 5 from Ad-Cre infection) after the passage and cell numbers were counted. The drawing shows the scheme of experiments. The mean±s.d. values from three replicates are shown. ( e ) Quantification of the BrdU incorporation of cells in d . Before harvest, BrdU was added for 20 min, and BrdU incorporation was analysed by FACS. The average FITC intensity of each cell population is shown in the graph. The mean±s.d. values from three replicates are shown. ( f ) Chromatin-enriched fractions of Ad-Cre treated or non-treated Claspin MEF (f/−) stably expressing the wild-type or DE/A mutant Claspin were analysed by western blotting to indicate proteins indicated and their phosphorylated forms.

Article Snippet: Anti-PCNA (sc-56, 1:200), anti-Pol α (sc-5920, 1:200), anti-LaminB (sc-6216, 1:200), anti-ATR (sc-1887 1:200), anti-Chk1 (sc-8408, 1:200) and anti-MCM2 (sc-9839, 1:200) antibodies were from Santa Cruz Biotechnology; anti-TopBP1 (A300-111A, 1:1,000) and anti-MCM2 S53 (A300-756A, 1:1,000) antibodies were from Bethyl; anti-Cdc7 (K0070-3, 1:1,000) and anti-Flag (M185-3L, 1:1,000) antibodies were from MBL; anti-Chk1 S345 (#2341, 1:1,000), anti-tubulin (T5168, 1:1,000), anti-myc (04362-34, 1:1,000) and anti-HA (16B12, 1:1,000) antibodies were from Cell Signaling, Sigma-Aldrich, Nacalai Tesque and Abcam, respectively.

Techniques: Mutagenesis, Western Blot, Purification, Clone Assay, Expressing, Infection, BrdU Incorporation Assay, Stable Transfection

Journal: Nature Communications

Article Title: Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells

doi: 10.1038/ncomms12135

Figure Lengend Snippet: ( a ) In vitro Cdc7-ASK kinase assays with wild-type and mutant Claspin proteins as substrates. Upper, schematic diagrams of Claspin derivatives including DE/A and other Claspin mutants in which serine/ threonine residues were replaced with alanine or glutamic acids; middle panel, autoradiogram; lower panel, CBB staining. Striped boxes indicate the segments in which serines/threonines were replaced with alanine or glutamic acid. ( b ) The wild-type or mutant Claspin proteins, as indicated, were coexpressed in 293T cells with Cdc7-ASK expression vectors. The whole-cell extracts were analysed by western blotting with anti-Flag antibody. Lanes 12–16, the samples were pretreated with lambda phosphatase before loading. ( c ) Wild-type (WT) and mutant Claspin proteins, as indicated, were expressed in 293T cells and were pulled down with M2 Flag beads. Associated proteins were analysed by western blotting to detect the proteins indicated. WT(HA) represents the HA-tagged wild-type Claspin used as a negative control. ( d ) The Flag-tagged N-terminal segment (#25) was coexpressed with the HA-tagged C-terminal polypeptide (#13) along with HA-tagged Cdc7 (wild-type or kinase dead ) and myc-tagged ASK and pulled down with M2 Flag beads. Association of #13 was analysed by western blotting using anti-HA antibody (lower panel). Arrowheads indicate the pulled down #13 polypeptide. *IgG; # Cdc7 polypeptide reacting with anti-HA antibody.

Article Snippet: Anti-PCNA (sc-56, 1:200), anti-Pol α (sc-5920, 1:200), anti-LaminB (sc-6216, 1:200), anti-ATR (sc-1887 1:200), anti-Chk1 (sc-8408, 1:200) and anti-MCM2 (sc-9839, 1:200) antibodies were from Santa Cruz Biotechnology; anti-TopBP1 (A300-111A, 1:1,000) and anti-MCM2 S53 (A300-756A, 1:1,000) antibodies were from Bethyl; anti-Cdc7 (K0070-3, 1:1,000) and anti-Flag (M185-3L, 1:1,000) antibodies were from MBL; anti-Chk1 S345 (#2341, 1:1,000), anti-tubulin (T5168, 1:1,000), anti-myc (04362-34, 1:1,000) and anti-HA (16B12, 1:1,000) antibodies were from Cell Signaling, Sigma-Aldrich, Nacalai Tesque and Abcam, respectively.

Techniques: In Vitro, Mutagenesis, Staining, Expressing, Western Blot, Negative Control