Figure 1.

TopBP1 fragments used in this study and their DNA binding properties. (A) Schematic of human TopBP1 and its fragments that were purified for structure–function experiments. The amino acid positions are indicated, and the eight boxes indicate the BRCT motifs. The ATR activating domain between BRCT domains 6 and 7 is indicated. (B) The GST-fusion proteins visualized by SDS–PAGE followed by Coomassie blue staining. (C) Preferential binding of TopBP1 and TopBP1 fragments to BPDE-damaged DNA. (Top panel) Unmodified (UM) or BPDE treated pUC19 plasmid DNAs (1 ng) which had been labeled with [γ-³²P]ATP were incubated with 3 pmol of full-length TopBP1 (FL) bound beads or beads carrying 3 pmol of the TopBP1-A, -B, -C or -D fragments in buffer containing 200 mM NaCl. The bound DNA was eluted by proteinase K, analyzed by agarose gel electrophoresis, and visualized by autoradiography. The input lanes contain fifty percent of DNA added to the reaction. The results from two experiments were quantified and plotted.