Figure 4.

S889 phosphorylation regulates SMARCAL1 ATPase activity. (A and B) U2OS cells were transfected with GFP-SMARCAL1 expression vectors and seeded in 96-well plates. Two days after transfection, the cells were fixed and stained with γH2AX antibodies and DAPI. Cells were imaged using an Opera automated confocal microscope and the GFP and γH2AX intensities per nucleus were measured using Columbus image analysis software. (A) The percentage of cells expressing GFP-SMARCAL1 with γH2AX intensity >1000 is graphed. Error bars are standard deviation (n = 7 wells for each of three biological replicates). ***P = 0.0008; **P = 0.003; *P = 0.04; two-tailed unpaired t-test comparing wild-type with each mutant. (B) The expression level as measured by GFP intensity is graphed. (C and D) The ATPase activity of Flag-SMARCAL1 purified from HEK293T cells was measured as a function of (C) fork DNA concentration or (D) SMARCAL1 concentration. The insets are representative immunoblots of the purified proteins used in the ATPase assays. Error bars are standard deviation (n = 3). In addition to a statistical difference between the overall curves, we calculated P-values for individual DNA or protein concentrations comparing wild-type with the mutant proteins. In (C) *P = 0.02; **P = 0.002; ***P = 0.0003; in (D) **P = 0.003; ***P < 0.0001. (E) The ATPase activity of Flag-SMARCAL1 proteins purified from baculovirus-infected insect cells in the presence of fork DNA was measured. **P = 0.0002, ***P < 0.0001. The inset is the Coomassie-stained gel of the purified proteins. (F) Flag-SMARCAL1 proteins purified from insect or human cells were immunoblotted with pS889 or total SMARCAL1 antibodies. The protein purified from human cells is slightly larger than the recombinant protein purified from insect cells due to extra amino acids encoded by the Gateway mammalian cell expression vector.