Figure 1.

SMARCAL1 acts at replication forks to prevent MUS81-catalyzed double-strand breaks. (A) Cells were labeled with EdU for 10 min, the EdU was removed, and thymidine was added for 20 min or HU was added for 3 h prior to purifying the nascent DNA–protein complexes using the iPOND procedure. (B) EdU-labeled cells were treated with 2 mM HU for the indicated lengths of time prior to performing iPOND. The “No Clk” controls in A and B are samples treated with EdU only, but no biotin-azide was added during the click reaction. (C) U2OS cells were transfected with siRNAs targeting SMARCAL1 (S), MUS81 (M), or nontargeting (NT) as indicated. Three days after transfection, the cells were either stained with antibodies to γH2AX or harvested for immunoblotting with the indicated antibodies. The percentage of cells staining positive for γH2AX was determined by immunofluorescent imaging from three independent experiments. Cells with >10 foci were counted as positive. Error bars are the standard deviation (SD; n = 3).