Dong Z , Tomkinson AE .

CA92584 NCI NIH HHS , R01 ES012512 NIEHS NIH HHS , P01 CA092584 NCI NIH HHS , ES012512 NIEHS NIH HHS , ES 012512 NIEHS NIH HHS

	Figure 1. DNA ligase IIIα is phosphorylated during cell cycle progression. (A) Human T24 cells were synchronized by density arrest. Aliquots of the culture were removed at the times indicated after replating at a lower density. The addition of nocodazole (Noc) blocked cells at G2/M. DNA ligase IIIα, Rb, (left panel) β-actin and XRCC1 (right panel) were detected in cell extracts by immunoblotting. The phosphorylation status of Rb serves as an indicator of cell cycle progression (23). (B) HeLa cell populations were enriched for the G1 (G1), G2/M (Noc) and S phases (DT) of the cell cycle as described in Materials and Methods section. DNA ligase IIIα was detected in the extracts by immunoblotting. (C) Cell extracts were prepared from asynchronous CHO cells (As) and CHO cells treated with hydroxyurea (Hu) and nocodazole (Noc). DNA ligase III was detected by immunoblotting. (D) Aliquots of an extract from an asynchronous population of T24 cells were incubated as indicated with λ phosphatase (λ) either in the presence (+) or absence (−) of the phosphatase inhibitor, NaF for 30 min at 37°C. DNA ligase IIIα was detected by immunoblotting. (E) DNA ligase IIIα was co-immunoprecipitated with XRCC1 antibody from an S phase HeLa cell extract. The immunoprecipitate was incubated with (+) or without (−) calf intestinal phosphatase (CIP) prior to the detection of DNA ligase IIIα by immunoblotting.
	Figure 2. Cdk2 phosphorylates DNA ligase III in vitro. (A) The consensus motif recognized by CDKs is shown with conserved putative CDK phosphorylation sites in mouse and human DNA ligase III below. (B) In the upper panel, Cdk6 and Cdk2 were immunoprecipitated from asynchronous HeLa cell extracts. In vitro kinase assays were performed with [γ-32P]ATP using an Rb fragment (Santa Cruz) or histone H1 (Sigma) as phosphorylation substrates. The protein kinase substrates were detected by Coomassie blue staining and phosphorimaging. In the lower panel, in vitro phosphorylation of DNA ligase III by Cdk6 and Cdk2 immunoprecipitated from asynchronous HeLa cell extracts was detected by phosphorimaging. (C) T24 cells density-arrested in G0 were released into the cell cycle and harvested at 6 h (G1) or 24 h (S). Cells were treated with nocodazole as described in Materials and Methods section to obtain G2/M enriched cells. In the upper and middle panels, Cdk2, Cdc2, cyclin A and cyclin B were detected in the cell extracts by immunoblotting. In the lower left panel, Cdk2 co-immunoprecipitated with cyclin A antibody (IP) from the indicated cell extract was detected by immunoblotting (IB). In the lower right panel, Cdc2 co-immunoprecipitated with cyclin B antibody (IP) from the indicated cell extract was detected by immunoblotting (IB). (D) Phosphorylation of histone H1 and DNA ligase III by CDK kinases immunoprecipitated with cyclin A (left panel) and cyclin B (right panel) antibodies from the indicated extracts. The protein kinase substrates were detected by Coomassie blue staining and phosphorimaging.
	Figure 3. Mapping the DNA ligase III sites phosphorylated in vitro by Cdk2. (A) The indicated fragments of DNA ligase III were expressed as GST fusion proteins. The grey and black boxes mark the positions of the N-terminal zinc finger and C-terminal BRCT domain, respectively. (B) Purified GST and the GST fusion proteins were incubated with immunoprecipitated Cdk2 and [γ-32P]ATP. (C) Wild type (wt) and mutant versions of DNA ligase III (S123/A and T104/A, S286/A), in which putative amino acid targets for Cdk2 phosphorylation were replaced with alanine, were incubated with immunoprecipitated Cdk2 and [γ-32P]ATP. After separation by SDS–PAGE, unlabeled proteins were visualized by Coomassie blue staining [upper panel in both (B) and C)] and labeleled proteins by phosphorimager analysis [lower panel in both (B) and (C)].
	Figure 4. Cdk2 phosphorylates DNA ligase III in vivo. (A) Proteins were immunoprecipitated with Cdk2, cyclin A or cyclin B antibodies from extracts of asynchronous HeLa cells. The immunoprecipitates were incubated with purified DNA ligase III and histone H1 in the presence of [γ-32P]ATP. DNA ligase III was detected by immunblotting with the DNA ligase III monoclonal antibody IF3 that recognizes unmodified and phosphorylated DNA ligase III and the antibody (α-phos-Ser123) raised against a DNA ligase III peptide (residues 116–130) encompassing phosphorylated Ser123. Purified histone H1 (Sigma) was detected by Coomassie blue staining. Labeled DNA ligase III and histone H1 were detected by phosphorimaging. (B) DNA ligase III from G1 (G1) and S (S) phase T24 cells was detected by immunoblotting with either 1F3 or α-phos-Ser123. (C) HeLa cells were transfected with expression vectors expressing HA-tagged versions of wild type (Cdk2-wt) and dominant negative cdk2(dn) (Cdk2-dn), and the empty expression vector (vector). HA-tagged Cdk2 and DNA ligase III were detected in cell extracts by immunoblotting.
	Figure 5. Oxidative damage induces dephosphorylation of DNA ligase IIIα. (A) DNA ligase III was detected in extracts from undamaged (con) S phase-enriched HeLa cells and from cells that had been either exposed to 10 Gy of ionizing radiation (IR) or 0.5% EMS (EMS) by immunoblotting. (B) Asynchronous HeLa cells were treated with the indicated concentrations of EMS and doses of IR. DNA ligase III in extracts from the damaged and undamaged (con) cell extracts was detected by immunoblotting with the antibody (α-phos-Ser123) raised against a DNA ligase III peptide (residues 116–130) encompassing phosphorylated Ser123. To control for protein loading, β-actin in the extracts was detected by immunoblotting. (C) Populations of HeLa cells enriched for S phase as described in Materials and Methods section were either mock treated or treated with 12 Gy of ionizing radiation (IR). DNA ligase III was detected in the cell extracts either directly by immunoblotting (upper panel) or by co-immunoprecipitation with an XRCC1 antibody followed by immunoblotting (lower panel). (D) S phase-enriched HeLa cells were treated with either hydrogen peroxide (H2O2, upper panel), menadione (MN, middle panel) or glucose oxidase (GOX, lower panel) as described in Material and Methods section. DNA ligase III was detected in cell extracts by immunoblotting.
	Figure 6. ATM mediates DNA ligase IIIα dephosphorylation induced by Oxidative damage. (A) S phase-enriched HeLa cells were treated with hydrogen peroxide (H2O2) either in the presence or absence of wortmannin as indicated. (B) ATM-deficient (pEBS7-FT) and ATM-complemented (pEBS7-YZ5) cell lines were either mock treated or treated with hydrogen peroxide (H2O2) as described in Materials and Methods section. DNA ligase III was detected in cell extracts by immunoblotting.
	Figure 7. Oxidative stress inhibits Cdk2 activity in an ATM-dependent manner. Cdk2 was immunoprecipitated from extracts of S phase-enriched HeLa cells, and ATM-deficient (pEBS7-FT) and ATM-complemented (pEBS7-YZ5) cells that had been treated with either hydrogen peroxide (H2O2) or EMS (EMS) as described in Materials and Methods. Immunoprecipitated Cdk2 was incubated with purified histone H1 (Sigma) and [γ-32P]ATP. After separation by SDS–PAGE, total histione H1 was visualized by Coomassie blue staining (H1, upper panel) and labeled histone H1 by phosphorimager analysis (32P, middle panel). Cdk2 protein in the immunoprecipitates was detected by imuunoblotting (lower panel, Cdk2).

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