The regulation of DNA replication is achieved by sequential assembly of different protein complexes to the replication origins. Among the currently known initiation proteins, the Origin Recognition Complex (ORC) is the initiator and serves as the landing pad for other initiation proteins. In order to identify unknown initiation proteins, we have performed a genetic screen to look for ORC-interacting proteins. One of the genes we found is the CDC48 gene....[ Read more ]

The regulation of DNA replication is achieved by sequential assembly of different protein complexes to the replication origins. Among the currently known initiation proteins, the Origin Recognition Complex (ORC) is the initiator and serves as the landing pad for other initiation proteins. In order to identify unknown initiation proteins, we have performed a genetic screen to look for ORC-interacting proteins. One of the genes we found is the CDC48 gene.

The budding yeast Cdc48p and its mammalian homologue p97 belong to the AAA (A̲TPase a̲ssociated with a variety of cellular a̲ctivities) super family and have previously been shown to participate in a number of important cellular activities. Our study is the first time to show that Cdc48p plays a role in DNA replication. In this study, in addition to the genetic interaction between ORC and CDC48, Cdc48p was shown to physically interact with ORC and Mcm proteins, suggesting that Cdc48p is a component of the pre-replicative complex (pre-RC). Furthermore, initiation of DNA replication was defective in cdc48-3 mutant cells. The defects were illustrated by a high plasmid loss rate which could be suppressed by tandem copies of replication origins on the plasmid, and by reduced frequency of initiation of DNA replication on chromosome in two-dimensional gel analysis.

In addition to its role in the initiation of DNA replication, Cdc48p was also shown in this study to be required for the execution of Start through degradation of the G1-CDK (cyclin-dependent kinase) inhibitor Far1p. We demonstrated that cdc48-td (temperature sensitive degron) mutant cells are defective for Start and arrest in G1 phase at the restrictive temperature with a low level of Cdc28p-Cln2p kinase activity and a high level of Far1p. The G1 arrest can be bypassed if FAR1 is deleted or CLN2 is expressed from a GAL promoter. Furthermore, cdc48-3 and cdc34-1 mutants have synthetic growth defect, consistent with Cdc48p’s participation in Far1p degradation through the Cdc34p-mediated ubiquitin-proteasome proteolysis pathway. Because previous conditional cdc48 mutants all arrest in G2/M at the restrictive temperatures, it was believed that Cdc48p plays an essential role(s) only during mitosis. Our studies uncovered a novel role of Cdc48p as a critical cell cycle regulator in G1 phase and shed new light on the regulation of Far1p in the cell cycle.

In this thesis, I also reported the progress of a novel phenotypic screen aimed at identification of novel initiation proteins. Tertiary screens were carried out to test possible rescue of high plasmid loss by 15 known replication initiation genes, among them 12 candidates have passed the primary and secondary screens. Three of the candidates passed the tertiary screen, and one of them had been cloned by complementation using a genomic library. The gene turned out to be CDT1, which has been recently reported as a gene encoding a replication initiation protein.