DNA damage signal activation in human cell-free extracts. (A) The duplex DNA substrates are blunt ended and 573 bp long. The gDNA contains a 68-nt single-stranded gap. (B) The gDNA is refractory to digestion with SpeI. (C) gDNA-specific phosphorylation of RPA32 and Chk1. Nuclear extracts were incubated without DNA (lane 1), with duplex DNA (lane 2), or with gDNA (lane 3). The indicated proteins were analyzed by Western blotting. (D) Proteins bound to biotinylated DNA were pulled down with streptavidin-coated beads and detected by Western blotting. The dsDNA and gDNA substrates are represented schematically. Biotin (black circles) and streptavidin-coated beads (dented gray circles) are shown.