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-C WT (lane 2), LacR-Claspin-C 3A (Thr⁹¹⁶, Ser⁹⁴⁵, Ser⁹⁸² to alanine) (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 (bottom 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 immunoblotting for phospho-Chk1 and Chk1 as indicated. The graph shows quantitative analysis of the data. C, LacO and control DNA by ethidium bromide staining after electrophoresis on a 0.8% agarose gel. 100, 200, 400 ng of LacO DNA (lanes 2–4) or control DNA (lanes 5–7), previously linearized by digestion with the single cutting restriction endonucleases SacI and BamHI, respectively, were loaded onto the gel. The plasmids were linearized to simplify visualization, but linearization does not affect ATR activation in our system.