Investigation on the mechanistic roles of Tom1 and Dia2 in DNA replication and cell cycle

Abstract

As cells duplicate and divide, the inheritance of accurate genetic information is critical to maintaining cell viability and preventing the generation of cancer-causing mutations. Thus, DNA replication is important and limited to once per cell cycle. The replication protein Cdc6 is required to assemble pre-replicative (pre-RC) complexes on origins of replication and is degraded after it has completed its function. The mechanisms of Cdc6 degradation in budding yeast are complex and not completely understood. I have identified a novel pathway that targets Cdc6 for degradation during G1 and at the G1 to S phase transition. The Hect E3 ligase Tom1 and the F-box protein Dia2 function co-operatively but independently of the SCFCdc4 complex. When this pathway is disrupted, the assembly of Cdc6 and Mcm4 on chromatin and replication origins is aberrant. Strikingly, defects in Tom1 or Cdc4 lead to increased DNA content in cells sensitized to re-replication. Therefore, multiple degradation pathways that limit Cdc6 protein levels cooperate to regulate pre-RC assembly and may act to prevent DNA re-replication. Preserving genomic stability is crucial to cell viability and proliferation. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle-dependent manner, but the identity of the ubiquitination pathway is unknown. I have uncovered that the Hect-domain E3 ligase Tom1 targets Dia2 for ubiqutin-dependent degradation during G1 and G2/M. Tom1 binding to Dia2 is switched during the cell cycle. Tom1 recognizes specific positively charged residues in a Dia2 degradation/NLS domain. Defects in Dia2 proteolysis such as the loss of positively charged residues and deletion of TOM1 cause a delay in G1 to S-phase progression. Together, these results suggest that Tom1 is required for degradation of Dia2 during the cell cycle and that Dia2 protein degradation contributes to G1 to S-phase progression.