The Hect-domain E3 ligase Tom1 and the F-box protein Dia2 control Cdc6 degradation in G1.

The accurate replication of genetic information is critical to maintaining chromosomal integrity. Cdc6 functions in the assembly of pre-replicative complexes and is specifically required to load the Mcm2-7 replicative helicase complex at replication origins. Cdc6 is targeted for protein degradation by multiple mechanisms in Saccharomyces cerevisiae, although only a single ...
pathway and E3 ubiquitin ligase for Cdc6 has been identified, the SCFCdc4 (Skp1/Cdc53/F-box protein) complex. Notably, Cdc6 is unstable during the G1 phase of the cell cycle, but the ubiquitination pathway has not been previously identified. Using a genetic approach, we identified two additional E3 ubiquitin ligase components required for Cdc6 degradation, the F-box protein Dia2 and the Hect-domain E3 Tom1. Both Dia2 and Tom1 control Cdc6 turnover during G1 phase of the cell cycle. Genetic epistasis analysis indicates that Dia2 and Tom1 function cooperatively but act separately from SCFCdc4. A mutant defective in all three ubiquitin ligase activities exhibits reduced viability. Tom1 and Dia2 each immunoprecipitate Cdc6, binding to a C-terminal region of the protein. Ubiquitination of Cdc6 is significantly reduced in dia2Δ and tom1Δ cells. Cdc6 and Mcm4 chromatin association is aberrant in tom1Δ and dia2Δ cells in G1 phase. Together, these results present evidence for a novel degradation pathway that controls Cdc6 turnover in G1 that may regulate pre-RC assembly.
J. Biol. Chem.
Date: Nov. 05, 2012
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