OVERPRODUCTION AND AFFINITY PURIFICATION OF SACCHAROMYCES-CEREVISIAE REPLICATION FACTOR-C

Yeast replication factor C (RF-C) is a heteropentamer encoded by the R FC1-5 genes. RF-C activity in yeast extracts was overproduced about 80 -fold after induction of a strain containing all five genes on a singl e plasmid, with expression of each gene placed under control of the ga lactose-inducible GAL1-10 promoter. This strongly indicates that overe xpression of the five known RFC genes is sufficient for overproduction of RF-C. Overexpression of all five genes was also necessary to achie ve overproduction of RF-C as omission of any single gene from the plas mid gave uninduced, i.e. normal cellular levels of RF-C. The interacti on between RF-C and proliferating cell nuclear antigen (PCNA) was stud ied with PCNA-agarose beads. Binding of RF C to PCNA-agarose beads is negligible in buffers containing 0.3 M NaCl. However, addition of Mg-A TP to the binding buffer caused strong binding of RF-C to the beads ev en at 0.8 M NaCl. Binding of ATP, but not its hydrolysis, was required for the strong binding mode as nonhydrolyzable analogs were also effe ctive. The existence of two distinct binding modes between PCNA and RF C was used as the key step in a greatly improved procedure for the pu rification of RF-C. RF-C from the overproduction strain purified by th is procedure was essentially homogeneous and had a severalfold higher specific activity than RF-C preparations that had previously been puri fied through multicolumn procedures.