THE CDC4 34/53 PATHWAY TARGETS CDC6P FOR PROTEOLYSIS IN BUDDING YEAST/

The budding yeast Cdc6 protein (Cdc6p) is essential for formation of p re-replicative complexes (pre-RCs) at origins of DNA replication. Regu lation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle, Cdc6p is normally only present at high levels during the G(1) phase of the cell cycle. This is partly be cause the CDC6 gene is only transcribed during G(1). In this article w e show that rapid degradation of Cdc6p also contributes to this period icity. Cdc6p degradation rates are regulated during the cell cycle, re aching a peak during late G(1)/early S phase. Removal of a 47-amino-ac id domain near the N-terminus of Cdc6p prevents degradation of Cdc6p. Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-me diated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p. A stable Cdc6p mutant which is no lo nger degraded by the Cdc4/34/53 pathway is, none the less, fully funct ional, Constitutive overexpression of either wild-type or stable Cdc6p does not induce rereplication and does not induce assembly of prerepl icative complexes after DNA replication is complete.