Journal: Journal of Biological Chemistry

Article Title: Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

doi: 10.1074/jbc.m111.237958

Figure Lengend Snippet: FIGURE 1. Tethering TopBP1 to DNA activates ATR phosphorylation of Chk1 in a defined system. A, 5 ng of purified LacR-TopBP1 (lane 1), LacR- Claspin-CWT(lane2),LacR-Claspin-C3A(Thr916,Ser945,Ser982toalanine)(lane 3), and RPA (lane 4) were fractionated by SDS-PAGE and then either stained with silver (top panels) or Western blotted with the indicated antibodies (bot- tom panels). B, LacR-TopBP1 (0, 0.125, 0.5, 2 nM) was added to kinase reactions containing 0.25 nM ATR and 12 nM His-Chk1. Reactions 1–4 contain no DNA, reactions 5–8 contain 3 pM LacO DNA plasmid, and reactions 9–12 contain 3 pM control DNA plasmid. ATR kinase activity was determined by immunoblot- ting for phospho-Chk1 and Chk1 as indicated. The graph shows quantitative analysis of the data. C, LacO and control DNA by ethidium bromide staining

Article Snippet: LacO plasmid is a 15-kb plasmid that contains 256 Lac operator repeats cloned into pBluescript (Addgene plasmid 17655, named Lac-I-SceI-Tet) (13).

Techniques: Phospho-proteomics, Purification, SDS Page, Staining, Western Blot, Plasmid Preparation, Control, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

doi: 10.1074/jbc.m111.237958

Figure Lengend Snippet: FIGURE 3. DNA binding by TopBP1 and Claspin is required for the syner- gistic stimulation of ATR phosphorylation of Chk1 in a defined system. A, LacR-TopBP1 and LacR-Claspin-C DNA binding in the presence or absence of IPTG is analyzed by EMSAs. The binding reactions contained 10 nM 32P- labeled 30-bp LacO DNA (lanes 1–3 and 6–11) or control DNA (lanes 4 and 5) and 50 nM LacR-Claspin-C (lanes 2, 3, and 5) or 5 nM, 10 nM, 20 nM LacR-TopBP1 (lanes 6–8 and 9–11). In lanes 3 and 9–11, the LacR fusion protein was prein- cubated on ice with IPTG at a final concentration of 150 M. The unbound DNA is indicated by a gray arrow, and the protein-bound DNA is indicated by a black arrow. The graph shows quantitative analysis of the data. B, ATR kinase reactions contained 0.125 nM LacR-TopBP1 and 6 nM LacR-Claspin-C and either no DNA (lanes 1 and 8), or 3, 12, or 48 pM LacO DNA (lanes 2–4 and 9–11), or 3, 12, or 48 pM control DNA (lanes 5–7 and 12–14) without (lanes 1–7) or with 150 M IPTG (lanes 8–14).

Article Snippet: LacO plasmid is a 15-kb plasmid that contains 256 Lac operator repeats cloned into pBluescript (Addgene plasmid 17655, named Lac-I-SceI-Tet) (13).

Techniques: Binding Assay, Phospho-proteomics, Labeling, Control, Concentration Assay