Expression of Cdc18/Cdc6 and Cdt1 during G(2) phase induces initiation of DNA replication

Cdc18/Cdc6 and Cdt1 are essential initiation factors for DNA replication. I n this paper we show that expression of Cdc18 in fission yeast G(2) cells i s sufficient to override the controls that ensure one S phase per cell cycl e. Cdc18 expression in G(2) induces DNA synthesis by re-firing replication origins and recruiting the MCM Cdc21 to chromatin in the presence of low le vels of Cdt1. However, when Cdt1 is expressed together with Cdc18 in G(2), cells undergo very rapid, uncontrolled DNA synthesis, accumulating DNA cont ents of 64C or more. Our data suggest that Cdt1 may potentiate re-replicati on by inducing origins to fire more persistently, possibly by stabilizing C dc18 on chromatin. In addition, low level expression of a mutant form of Cd c18 that cannot be phosphorylated by cyclin-dependent kinases is not suffic ient to induce replication in G(2), but does so only when co-expressed with Cdt1. Thus, regulation of both Cdc18 and Cdt1 in G(2) plays a crucial role in preventing the re-initiation of DNA synthesis until the next cell cycle .