Fig 2.

The inter-BRCT region of Dpb11 (aa 253 to 290) is a GINS-interacting domain. (A) Schematic drawings of the Dpb11 constructs. The numbers placed above the drawings show the positions of amino acids. The BRCT motif is shown as an open box. (B) Yeast two-hybrid assay. Cells harboring the plasmids indicated were grown on SC plates lacking leucine and tryptophan, lifted with filter paper, and subjected to a β-galactosidase assay. AD, activation domain; DB, DNA binding. (C) Immunoprecipitation (IP) was performed as described in Materials and Methods. Samples were analyzed by Western blotting. Gal4AD-HA-Dpb11 aa 253 to 290 and GINS proteins were detected with anti-HA and anti-GINS antibodies, respectively. The ratio of the amounts of samples loaded on the gel was 1:10:1 (Input/IP/Sup. [supernatant]). *, nonspecific signals; Vec., vector. (D) The in vitro pulldown assay was performed as described in Materials and Methods. Samples were analyzed by Western blotting. GST-Dpb11 and GINS proteins were detected with anti-GST and anti-GINS antibodies, respectively. The ratio of the amount of samples loaded on the gel was 1:2:1 (Input/Beads ppt. [precipitates]/Sup.). (E) Low-copy-number vector (Vector) and low-copy-number DPB11 constructs (YCp-DPB11: wild-type DPB11 [Wt] and dpb11Δ253 to 290 [Δ253–290]) were introduced into YST756 (Δdpb11 [YEp–DPB11]), serially diluted, and grown on an SC plate lacking leucine and tryptophan (SC - Leu, Trp) or on an SC plate containing 5-fluoroorotic acid (5-FOA) (SC - Leu, Trp + FOA) to eliminate the YEp-DPB11 plasmid from the cells.