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. 2015 Feb 6;290(12):7586–7601. doi: 10.1074/jbc.M115.640383

FIGURE 6.

FIGURE 6.

A Dpb11 mutant (Dpb11-m1,m2,m3,m5,ΔCTD) does not bind ssDNA but it binds Sld2T84D, Mcm2-7, and Cdc45 like wild type Dpb11. A, charge reversal mutations in the BRCT 4 region of Dpb11 (amino acids 430–615) that were studied for interaction with ssARS1–3. B, various concentrations of radiolabeled full-length PKA·Dpb11-wild-type, dpb11-m1, dpb11-m2, dpb11-m3, dpb11-m4 (not shown, binds like Dpb11·wild-type), dpb11-m5, or dpb11-m6 (not shown, binds like Dpb11·wild-type) were incubated with 4 pmol of ssARS1–3 in a biotin pulldown assay. The beads were washed and analyzed as described under “Experimental Procedures.” The results from the experiment were quantified, averaged, and plotted as picomoles of Dpb11 bound versus picomoles of input Dpb11. C, different concentrations of PKA-Dpb11 and PKA·dpb11-m1,m2,m3,m5,ΔCTD were incubated with 4 pmol of ssARS1–3 in a biotin pulldown assay. The beads were washed and analyzed as described under “Experimental Procedures.” D, results from experiments similar to C were quantified, averaged, and plotted as picomoles of Dpb11 bound versus picomoles of Dpb11 input. E, different concentrations of radiolabeled PKA-Sld2T84D were incubated with 30 pmol of GST-Dpb11 wild-type, GST·dpb11-m1,m2,m3,m5,ΔCTD, or GST tag alone in a GST pulldown assay as described under “Experimental Procedures.” The bound radioactive Sld2T84D was analyzed by SDS/PAGE followed by phosphorimaging. These results were then quantified, averaged, and plotted as picomoles of Sld2T84D bound versus picomoles of Sld2T84D input. F, different concentrations of radiolabeled PKA·Mcm2-7 was incubated with 10 pmol of GST-Dpb11 wild-type, GST·dpb11-m1,m2,m3,m5,ΔCTD, or GST tag alone in a GST pulldown assay as described under “Experimental Procedures.” The bound radioactive Mcm2-7 was analyzed by SDS/PAGE followed by phosphorimaging. These results were then quantified, averaged, and plotted as picomoles of Mcm2-7 bound versus picomoles of Mcm2-7 input. G, different concentrations of radiolabeled PKA-Cdc45 was incubated with 10 pmol of GST-Dpb11 wild-type, GST·dpb11-m1,m2,m3,m5,ΔCTD, or GST tag alone in a GST pulldown assay as described under “Experimental Procedures.” The bound radioactive Cdc45 was analyzed by SDS/PAGE followed by phosphorimaging. These results were then quantified, averaged, and plotted as picomoles of Cdc45 bound versus picomoles of Cdc45 input.