Redundant control of rereplication in fission yeast

The initiation of DNA replication at replication origins in eukaryotic cell s is tightly controlled to ensure that the genome is duplicated only once e ach cell cycle. We present evidence that in fission yeast, independent regu lation of two essential components of the initiation complex, Cdc18 and Cdt 1, contributes to the prevention of reinitiation of DNA replication. Cdc18 is negatively controlled by cyclin-dependent kinase (CDK) phosphorylation, but low level expression of a mutant form of Cdc18 lacking CDK phosphorylat ion sites (Cdc18(CDK)) is not sufficient to induce rereplication. Similar t o Cdc18, Cdt1 is expressed periodically in the cell cycle, accumulating in the nucleus in G(1) and declining in G(2). When Cdt1 is expressed constitut ively from an ectopic promoter, it accumulates in the nucleus throughout th e cell cycle but does not promote reinitiation. However, constitutive expre ssion of Cdt1, together with Cdc18(CDK), is sufficient to induce extra roun ds of DNA replication in the absence of mitosis. Significantly greater leve ls of rereplication can be induced by coexpression of Cdc18CDK and a Cdt1 m utant lacking a conserved C-terminal motif. In contrast, uncontrolled DNA r eplication does not occur when either mutant protein is expressed in the ab sence of the other. Constitutive expression of wild-type or mutant Cdt1 als o leads to an increase in the levels of Cdc18(CDK), possibly as a result of increased protein stability. Our data are consistent with the hypothesis t hat control of rereplication depends on a redundant mechanism in which nega tive regulation of Cdt1 functions in parallel with the negative regulation of Cdc18.