DNA replication is a critical event in the cell cycle, in the development of organisms, and in cancer. Eukaryotic DNA replication has been most extensively studied in the budding yeast S. cerevisiae because it has a small genome, powerful genetics and the ability to maintain plasmids to provide a replication assay. While the DNA elements and many proteins involved in the initiation of yeast DNA replication have been identified, some replication initiation proteins, including the crucial replicative helicase responsible for unwinding DNA double helix, have not been discovered....[ Read more ]

DNA replication is a critical event in the cell cycle, in the development of organisms, and in cancer. Eukaryotic DNA replication has been most extensively studied in the budding yeast S. cerevisiae because it has a small genome, powerful genetics and the ability to maintain plasmids to provide a replication assay. While the DNA elements and many proteins involved in the initiation of yeast DNA replication have been identified, some replication initiation proteins, including the crucial replicative helicase responsible for unwinding DNA double helix, have not been discovered.

We have performed a genetic screen to identify currently unknown initiation proteins for yeast DNA replication. This comprehensive phenotypic screen is a novel adaptation of several genetic strategies. A primary screen based on plasmid loss was used to select mutants with defects in plasmid replication and/or segregation. A secondary screen with a plasmid containing multiple ARS (autonomously replicating sequence) was designed to identify replication initiation mutants among the primary screen candidates. An (ade2 ade3)-based yeast colony color assay was employed to make this large-scale screen feasible and relatively easy. These strategies have been successfully tested and the primary and secondary screens have been finished. From about 30,000 EMS mutagenized yeast strains, 182 candidates with defects in the initiation of DNA replication have been isolated. In addition, 51 mutant strains with plasmid segregation or other defects than replication initiation have also been identified. A tertiary screen has been partly accomplished by using known replication genes to test potential complementation of the initiation mutants and thus excluding mutants in the known genes in order to expedite cloning of previously unknown replication initiation genes.

Twenty-eight of the initiation mutant strains were also temperature sensitive (ts) for growth, and nineteen of them have passed the tertiary screens so that they are likely mutants in unknown initiation genes. Furthermore, a previously uncharacterized gene, YAE1 (Yet Another Essential gene), has been found to rescue both the ts lethal and high plasmid loss phenotypes of one of the ts mutants. Therefore, Yae1p is most likely a previously unknown replication initiation protein. Discovery of initiation proteins will help to advance the field of yeast DNA replication and facilitate studies of DNA replication in higher eukaryotes in both normal and cancerous cells.