The duplication of a single cell involves a series of tightly controlled stages called the cell cycle. The faithful replication of genetic information is essential to cell division as disruption of DMA replication leads to genomic instability, a hallmark of cancer cells. Ubiquitination is one of the key regulators of cell cycle progression. Members of the highly conserved, modular SCF (Skp1/Cdc53/F-box protein) ubiquitin ligase complexes are important mediators of ubiquitin-dependent regulation of cell division. F-box proteins serve as substrate-specific adaptors for SCF complexes. Many F-box proteins have been identified in both humans and model eukaryotic systems and mutation of SCF family members contributes to tumorigenesis. This implies that ubiquitin-mediated destruction of key cell cycle proteins is an important mechanism for controlling cell proliferation. The specific objective of this proposal is to determine the role of the F-box protein Dia2 in the regulation of DMA replication. Dia2 is a chromatin-bound protein that binds replication origins in a cell cycle dependent manner. Preliminary data indicate that Dia2 forms complexes with DMA replication proteins, including the MCM2-7 replicative helicase and Cdc17, a subunit of DMA polymerase a/primase complex. The Dia2 protein is targeted for destruction during G1, a time when replication complexes are assembled. Interestingly, dia2 null and F-box deletion mutants are hypersensitive to DMA damaging agents and accumulate DMA damage foci, suggesting that a SCFDia2 complex plays a role in maintaining genomic stability. The central hypothesis is that Dia2 functions as part of an SCF ubiquitin ligase that regulates DNA replication. In the absence of this function, the genome is unstable. In addition, proteolysis of the Dia2 protein may act as a switch to control SCFDia2 activity in the regulation of DNA replication. The specific goals of this proposal are to: 1. Examine the regulation of Dia2 and its effects on DNA replication by determining how Dia2 is recruited to replication origins and how Dia2 proteolysis is controlled, and 2. Identify the role of Dia2 protein complexes in DNA replication by determining their composition and examining whether they are cell cycle regulated. SCFDia2 protein targets will be identified by a candidate approach and continued development of a genetic method for isolating substrates. Relevance: This project examines biochemical processes that control normal cell growth and proliferation. When defective, these processes contribute to tumor formation. The goal is to understand how the processes work in normal cells so that anti-cancer therapies can be developed in the future.
