The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediat
ed proteolysis by the E3 ubiquitin ligase SCFCDC4 from the end of G(1) phas
e until mitosis in the budding yeast Saccharomyces cerevisiae. Here we desc
ribe a dominant-negative CDC6 mutant that, when overexpressed, arrests the
cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevent
s passage through mitosis. This mutant protein inhibits CDKs more efficient
ly than wild-type Cdc6, in part because it is completely refractory to SCFC
DC4- mediated proteolysis late in the cell cycle and consequently accumulat
es to high levels. The mutation responsible for this phenotype destroys a p
utative CDK phosphorylation site near the middle of the Cdc6 primary amino
acid sequence. We show that this site lies within a novel Cdc4-interacting
domain distinct from a Cdc4-interacting site identified previously near the
N-terminus of the protein. We show that both sites can target Cdc6 for pro
teolysis in late G(1)/early S phase whilst only the newly identified site c
an target Cdc6 for proteolysis during mitosis.