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. 2011 Apr 18;286(22):19229–19236. doi: 10.1074/jbc.M111.237958

FIGURE 2.

FIGURE 2.

TopBP1 and Claspin function synergistically to stimulate ATR phosphorylation of Chk1 when tethered to DNA in a defined system. A, ATR kinase reactions contained no mediator proteins (lanes 1–3), 0.125 nm LacR-TopBP1 (lanes 4–6), 6 nm LacR-Claspin-C (lanes 10–12), or both (lanes 7–9). Reactions 1, 4, 7, and 10 contain no DNA (−), reactions 2, 5, 8, and 11 contain 3 pm LacO DNA (L), and reactions 3, 6, 9, and 12 contain 3 pm control DNA (C). B, GST-TopBP1 is not synergistic with LacR-Claspin, indicating that the mediators must co-localize to stimulate ATR effectively. ATR kinase reactions contained 6 nm LacR-Claspin-C and either 3 pm LacO DNA (lanes 1–7) or 3 pm control DNA (lanes 8–14), and either no TopBP1 (lanes 1 and 8), 0.0417, 0.125, or 0.375 nm LacR-TopBP1 (lanes 2–4 and 9–11), or 0.417, 1.25, or 3.75 nm GST-TopBP1 (lanes 5–7 and 12–14). C, mutations in the Chk1 binding domain of LacR-Claspin-C (Thr916, Ser945, Ser982 changed to alanine) abolish its ability to synergize with LacR-TopBP1 to activate ATR phosphorylation of Chk1. ATR kinase reactions contained 0.125 nm LacR-TopBP1, 3 pm LacO DNA (lanes 1–7), or 3 pm control DNA (lanes 8–14), and either no LacR-Claspin (lanes 1 and 8), 0.7, 2, or 6 nm LacR-Claspin-C WT (lanes 2–4 and 9–11), or mutant (3A, lanes 5–7 and 12–14).