Purification of the cdc8 protein of Saccharomyces cerevisiae by complementation in an aphidicolin-sensitive in vitro DNA replication system.

Abstract

DNA synthesis in vitro in Brij-treated Saccharomyces cerevisiae requires the product of the CDC8 gene (Hereford, L. M. & Hartwell, L. H. (1971) Nature (London) New Biol. 234, 171-172). Extracts of wild-type A364a yeast restore DNA synthesis in Brij-treated cdc8, a mutant containing a thermolabile cdc8 gene product. This constitutes a complementation assay by which the cdc8 gene product can be monitored during purification. A heat-stable protein responsible for this complementation has been partially purified from both wild-type A364a cells and from a cdc8 temperature-sensitive mutant. The complementation activity from the mutant is thermolabile when compared to the wild-type activity, indicating that CDC8 is the structural gene for the protein.