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

FIGURE 2.

FIGURE 2.

Dpb11 binds to T-rich ssDNA of 40 nucleotides. A, oligonucleotide sequences used. B, purified GST-Dpb11 or GST were studied for interaction with radiolabeled single stranded (ss) or double stranded (ds) sequences from the ARS1 origin in budding yeast. 13 pmol of GST-Dpb11 was incubated with DNA and glutathione-Sepharose for 10 min at 30 °C. The glutathione-Sepharose beads were washed and analyzed as described under “Experimental Procedures.” The results from the experiment were quantified, averaged, and plotted as picomoles of DNA bound versus picomoles of input DNA. C and D, PKA-Dpb11 was radiolabeled and used in a biotin pulldown assay. Single-stranded or double-stranded DNA from yeast origins (ARS1 or ARS305) with a 3′ biotin tag was conjugated to magnetic streptavidin beads. 4 pmol of this biotinylated DNA was incubated with radiolabeled PKA-Dpb11 at 30 °C for 10 min. As a control, beads without DNA were incubated with PKA-Dpb11. 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 the picomoles of input Dpb11. E, GST Dpb11 or GST was studied for interaction with radiolabeled DNA (ssARS1–3 or modifications of ssARS1–3) as indicated in the graph. 13 pmol of GST Dpb11 or GST was incubated with glutathione-Sepharose and varying amounts of DNA at 30 °C for 10 min. The glutathione-Sepharose beads were washed and analyzed as described under “Experimental Procedures.” The results from the experiment were quantified, averaged, and plotted as picomoles of DNA bound versus picomoles of input DNA. F, PKA-Dpb11 was radiolabeled and used in a biotin pulldown assay. Single-stranded DNA of varying lengths (ss80dT, ss60dT, ss40dT, and ss20dT) with a 3′ biotin tag was conjugated to magnetic streptavidin beads. 4 pmol of this biotinylated DNA was incubated with radiolabeled PKA-Dpb11 at 30 °C for 10 min. As a control, beads without DNA were incubated with PKA-Dpb11. 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. G, similar to F, except matching nucleotides instead of matching picomoles of DNA. Therefore, 4 pmol of ss80dT, 5.3 pmol of ss60 dT, 8 pmol of ss40dT, or 16 pmol of ss20dT were used in the pulldowns.