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

FIGURE 3.

FIGURE 3.

DNA binding by TopBP1 and Claspin is required for the synergistic 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 preincubated on ice with IPTG at a final concentration of 150 μm. 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 μm IPTG (lanes 8–14).