Yeast 2-microns plasmid DNA replication in vitro: purification of the CDC8 gene product by complementation assay

Extracts of the yeast Saccharomyces cerevisiae support DNA replication on exogenous yeast 2-microns plasmid DNA templates. A crude extract from a S. cerevisiae cell division cycle mutant, cdc8-1, expressed the temperature-sensitive phenotype since it could be inactivated at 42 degrees C in vitro. This heat-inactivated extract was fully complemented by the addition of either wild-type or cdc8-1 single-stranded DNA binding protein (SSB). restoration by SSB of the activity of the mutant cell extract allowed replication like that of a wild-type crude extract, as shown by bidirectional DNA synthesis from the in vivo origin. The DNA binding protein specifically stimulates the reaction catalyzed by yeast DNA polymerase I, a true DNA replicase, using the hybrid of phi X174 single-stranded DNA and a restriction endonuclease fragment as a template. It also increases processivity of DNA polymerase I at least 10-fold. Escherichia coli SSB, but not T4 gene 32 protein, can substitute for yeast SSB. Both restoration of DNA synthesis in the heated mutant cell extract and stimulation of the DNA polymerase I reaction by SSB from cdc8-1 cells are inactivated at nonpermissive temperatures, suggesting that yeast SSB is the CDC8 gene product.