Yeast 2-micrometer plasmid DNA replication in vitro: origin and direction.

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Abstract
Most yeast strains harbor extrachromosomal 2-.mu.m DNA, and this DNA synthesis, like nuclear DNA replication, is strictly under cell cycle control. A soluble extract of yeast S. cerevisiae carries out semiconservative replication of added 2-.mu.m DNA and Escherichia coli chimeric plasmids containing the 2-.mu.m DNA. Replication is initiated on 10% of the DNA, and 1 round of replication is completed. The major products in early stages of replication are .theta. ("eye") forms which originate 140 .+-. 50 nucleotides within 1 of 599 base-pair inverted repeats of 2-.mu.m DNA. Their replication is bidirectional and discontinuous. Extracts prepared from the cell division cycle mutant cdc8 show temperature-sensitive 2-.mu.m DNA synthesis in vitro, suggesting that this in vitro system resembles in vivo 2-.mu.m plasmid DNA replication. This system should provide a useful assay for the purification and characterization of yeast DNA replication proteins.