The human licensing factor for DNA replication Cdt1 accumulates in G(1) and is destabilized after initiation of S-phase

S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in Gl, and passage through S- phase removes the license to replicate. In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring that replicati on occurs once per cell cycle. We show that the human Cdt1 homologue (hCdt1 ), a nuclear protein, is present only during G(1). After S-phase onset, hCd t1 levels decrease, and it is hardly detected in cells in early S-phase or G(2). hCdt1 can associate with the DNA replication inhibitor Geminin, howev er these two proteins are mostly expressed at different cell cycle stages. hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase-arrested cells, and its levels do not change dramatically during a cell cycle, sugge sting that proteolytic rather than transcriptional controls ensure the time ly accumulation of hCdt1. Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase. In contrast, hCdc6/18 levels are constant throu gh most of the cell cycle and are only low for a brief period at the end of mitosis. These results suggest that the presence of active hCdt1 may be cr ucial for determining when licensing is legitimate in human cells.