FIGURE 3.

Deletion analysis of TopBP1 binding to RPA-ssDNA. A, schematic depicting the relative positions of BRCT domains (numbered) within the TopBP1 protein. B–G, Myc-tagged TopBP1 derivatives corresponding to the indicated BRCT domain(s) were produced by IVT and then mixed with biotin-linked ssDNA (1.5 kb) and binding buffer. Recombinant RPA was optionally added as indicated. After incubation, the ssDNAs were isolated using streptavidin magnetic beads, washed, and eluted, and bound proteins were detected by Western blot. The TopBP1 proteins were detected using mAb 9E10, which recognizes the myc tag. Input refers to 1.6% of the initial binding reaction. Data shown for each experiment are representative of a minimum of two independent biological replicates.