Single copy replication control is regulated by genes involved in ubiquitin metabolism in the yeast Saccharomyces cerevisiae

DNA replication is limited to only once per cell cycle in eukaryotic cells. A model proposed by Ron Laskey and Julian Blow to explain how cells accomplish this is called the licensing factor model. In this model they propose the existence of an entity that must be present to allow DNA to be replicated, and that is inactivated until the next cell cycle by the passing DNA replication fork. In order to identify potential inhibitors of re-replication a genetic screen was developed in the yeast Saccharomyces cerevisiae that is based on the idea that if a cell contained a temperature sensitive allele of such an inhibitor it would make too much DNA at the restrictive temperature and that this would be lethal. This lethality should, however, be circumvented by a second mutation in DNA polymerase that blocks DNA synthesis at the high temperature. A mutation was identified using this screen that was sensitive to high copy numbers of the POL1 gene but was rescued by a temperature sensitive allele of this gene, cdc17-1. Cells with this mutation also have elevated DNA contents including cells with greater than a 2C DNA content; thus the mutation was named dos1-1 for DNA over synthesis. The mutation was found to be a single base deletion in a gene called DOA4 which encodes a ubiquitin hydrolase, an enzyme involved in the ubiquitin dependent proteolytic pathway. Further analysis of this mutation revealed that it displays many of phenotypes seen in mutations that disturb DNA metabolism such as elevated chromosome loss and a large percentage of large budded cells with a single nucleus at the neck of the bud. Analysis of ubiquitin pools revealed that at high temperature the cells were deficient in free ubiquitin. Consistent with this, another gene involved in ubiquitin metabolism, UBI4, was identified as a single copy suppressor of the temperature sensitivity of dos1-1. In addition, double mutants lacking both DOA4 and UBI4 have decreased viability which implies that they are involved in parallel pathways essential for growth. The data reported in this thesis indicate a direct link between single copy control of DNA replication in eukaryotic cells and ubiquitin metabolism.