Figure 2

Direct interaction between recombinant Pol1 (278–512) and Cdc27 proteins. A. Expression of H6-Pol1 (278–512) in E. coli detected by immunoblotting using anti-MRGS antibodies. Total protein extracts prepared from cells carrying the control plasmid pQE32-Pol1 (278–512)-REV (lanes 1, 2), in which the relevant pol1⁺ sequence is present in the wrong orientation, or pQE32-Pol1 (278–512) (lanes 3, 4), either before (lanes 1, 3) or after (lanes 2, 4) induction of protein expression by addition of IPTG. The position of the 25 kDa marker is shown. B. Purified GST and GST – Cdc27 (273–352) proteins detected by Coomassie staining following SDS-PAGE. C. Binding assays using GST – Cdc27 fusion proteins. Bound H6-Pol1 was detected by Western blotting as in part A. Lanes 1, 3, 5: binding of H6-Pol1 to GST. Lanes 2, 4, 6: binding of binding of H6-Pol1 to GST – Cdc27 (273–352). In each case the bound fraction corresponds to approximately 15–20% of the input. Lanes 1 and 2: assay performed in PBS containing 0.1% Triton X100; lanes 3 and 4: PBS containing 0.25% Triton X100; lanes 5 and 6: PBS containing 0.5% Triton X100. In the absence of detergent Pol1 binds equally well to both GST and GST-Cdc27 (273–352) proteins.