Hou et al. 10.1073/pnas.0503525102. |
Supporting Materials and Methods
Supporting Figure 3
Supporting Figure 4
Supporting Figure 3
Fig. 3. Functional analysis of wild-type and mutant Sir1p origin recognition complex (ORC) interaction region (OIR)*/Orc1p-bromo-adjacent homology (BAH) complexes. (A) Data from size-exclusion chromatography of wild-type His-6-OIR* (1) and mutant His-6-OIR*Y489A (2) (magenta trace labeled OIR*), Orc1p-BAH (gold trace labeled Orc1p-BAH), and an equal molar mixture of the relevant His-6-OIR*/Orc1p-BAH mixture (blue trace labeled Orc1p-BAH-OIR*). Each protein domain or complex (30 m M) was analyzed in independent experiments using the same S-100 column; the column was washed extensively between analysis of each protein or protein mixture. The peaks represent the fractions at which the protein domain or complex eluted, as indicated in the figure and measured by UV absorption and SDS/PAGE analysis. (B) Data from Ni-affinity chromatography used to measure the interaction between wild-type and indicated mutant His-6-OIR* proteins and the Orc1p-BAH domain. The starting mixture (lanes 1, 5, and 9), the protein that failed to bind the column (lanes 2, 3, 6, 7, 10, and 11) and the protein that bound the resin (lanes 4, 8, and 12) are shown for the each experiment with wild-type His-6-OIR* (lanes 1–4), His-6-OIR* Y489A (lanes 5–8) and His-6-OIR*L504A (lanes 9–12). In each experiment, the OIR* contains a His-6 tag, but the Orc1p-BAH domain does not.
Fig. 4.
Surface electrostatics of the origin recognition complex (ORC) interaction region (OIR)*/bromo-adjacent homology (BAH) interface. Diagrams of the Orc1 BAH domain (Left) and Sir1 OIR domain (Right) are shown as in Fig. 2D with interacting residues indicated with connecting lines. The surface potential is displayed for each domain at ± 8 kBT/e for positive (blue) or negative (red) charge potential using the program GRASP (1).1. Nicholls, A., Sharp, K. A. & Honig, B. (1991) Proteins 11, 281–296.
Supporting Materials and Methods
His-6-OIR* and two point mutant variants of the domain (His-6-OIR*-Y489A and His-6-OIR*-L504A) were purified by Ni-nitrilotriacetic acid (NTA) chromatography followed by size-exclusion chromatography as described in Materials and Methods. Each of the His-6-tagged fusions was mixed with purified Orc1p bromo-adjacent homology (BAH) domain that lacked a His-6 tag at a final concentration of ≈30 mM for each protein in S-100 buffer, and the mixture was incubated on ice for 30 min.
For gel filtration analysis of the complex, 2 ml each of the His-6-OIR*/BAH mixtures were fractionated over a Sephacryl S-100 column equilibrated with S-100 buffer into 3-ml fractions. Before analysis of the complex, similar concentrations of the individual domains were fractionated over the same column to serve as references. The column was washed extensively between samples. Fifteen microliters of each fraction was analyzed by 15% SDS/PAGE to confirm the identity of each peak.
For Ni-NTA affinity chromatography, 2 ml of each of the above His-6-OIR*/BAH mixtures was loaded onto a 0.5 ml Ni-NTA column equilibrated in lysis buffer. The columns were washed with 2 ml lysis buffer and bound proteins were eluted with 2 ml elution buffer. Five microliters of each fraction was analyzed by 15% SDS/PAGE.