Activation of DNA damage checkpoint pathways prevents the binding of RecQL4, Mcm10, and Ctf4 to replication origins. (A) HeLa cells were irradiated with UV (10 or 20 J/m2), treated with 2 μg/ml 4NQO (4N) or 200 μg/ml zeocin (Ze) for 3 hr. The level of indicated protein in the soluble (SF) or chromatin (CF) fraction was examined by Western blotting. Lane M represented the mock-treated control. (B) The 293T cells stably expressing FLAG-RecQL4 proteins were irradiated with 20 J/m2 UV, and incubated either in the absence or presence of 5 mM caffeine for 3 hr. After preparation of cell extracts, immunoprecipitations were carried out using anti-FLAG M2 agarose beads. FLAG peptide (0.2 mg/ml) was added to whole cell extracts for control immunoprecipitations (Con). (C) HeLa cells irradiated with 20 J/m2 UV were incubated either in the absence or presence of 5 mM caffeine for 3 hr, and the binding of RecQL4, Mcm10, and Ctf4 to replication origins in the Mcm4 upstream promoter region (M4 Ori) or the β-globin locus (β-globin Ori) was examined by ChIP analysis. The primer sets for the 4 kb distal region of M4 Ori and the 9 kb distal region of β-globin Ori were used as internal controls in PCR analyses.