Figure 6.

Sld2T84D enhances the ability of ssARS1-1 to pulldown Dpb11. (A) A direct interaction between Sld2T84D and Dpb11 was studied. GST–Dpb11 or GST (40 pmol) and varying amounts of Sld2T84D, as indicated in the figure, were mixed with 1.3 mg glutathione agarose beads and incubated for 5 min at 30°C. The beads were washed and analyzed as described in ‘Materials and Methods’ section. The results from experiments similar to (A) were quantified and plotted as the fraction of Sld2T84D bound versus the pmol input of Sld2T84D (B). (C) The interaction of Sld2T84D and Dpb11 was studied in the presence of excess ssARS1-1. The experiment was performed as described above with the addition of increasing amounts of ssARS1-1. The results from experiments similar to (C) were quantified and plotted as the fraction Sld2T84D bound versus the ratio of ssARS1-1 and Sld2T84D (D). Adding excess ssARS1-1 slightly stimulates GST-Dpb11 pulldown of Sld2T84D. (E) Dpb11 (1.27 or 4.75 pmol) and increasing amounts of Sld2T84D, as indicated in the figure, were mixed with 7 pmol biotinylated ssARS1-1 conjugated to 4.3 × 10⁵ bead/µl streptavidin agarose and incubated for 5 min at 30°C. The beads were washed and analyzed as described in ‘Materials and Methods’ section. The results from experiments similar to (E) were quantified and plotted as the pmol of Dpb11 bound versus the pmol of Sld2T84D input (F). In the absence of Sld2T84D, Dpb11 binds weakly to ssARS1-1, and the addition of Sld2T84D enhances the amount of Dpb11 pulled down by ssARS1-1.