Fig. 1. A simple model describing how DNA replication is limited to once per cell cycle in budding yeast

ONCE PER CELL CYCLE REPLICATION

Eukaryotic chromosomes are replicated from large numbers of replication origins during a discrete phase of the cell cycle known as the synthetic or S phase. To ensure that the entire genome is precisely duplicated during the cell cycle it is essential both that a sufficient number of origins are activated and that no origin is used more than once in a cell cycle. The key to understanding how this is accomplished lies in the regulation of the prereplicative complex ( pre-RC - ref 3), an assemblage of proteins essential for DNA replication. In all systems examined, the assembly of the pre-RC (a step called licensing) can only occur during a short window of time prior to S phase.

Pre-RC assembly in budding yeast is restricted to early in the G1 phase of the cell cycle. This is because cyclin dependent kinases (CDKs), which are activated late in G1 phase and remain active until the end of mitosis, prevent licensing (see Fig. 1 and ref. 36). Inactivation of CDKs, for example, during G2 phase, is sufficient to cause the inappropriate re-licensing of replication origins ( 5, 19)

Over the past decade, we have learned that CDKs prevent pre-RC assembly directly, inhibiting each pre-RC component by a different mechanism (see Fig. 2). One nice example is the Cdc6 protein. Phosphorylation of Cdc6 in late G1 phase by the G1 cyclins (Cln1,2) targets Cdc6 for very rapid proteolysis that is mediated by the E3 ubiquitin ligase, SCF ( Lucy Drury). It is the Cdc4 subunit of SCF interacts directly with the phosphorylated Cdc6 protein ( 16, 26). Later in the cell cycle (e.g. during G2/M), Cdc6 becomes more stable, but is still inactive ( 20). Satoru Mimura has recently shown that the binding of the mitotic cyclin Clb2 to Cdc6 inactivates Cdc6 during G2/M.  Interestingly, Satoru has shown that Clb2 binds specifically to a phosphopeptide motif in the N-terminus of Cdc6 (40).  This novel phosphpeptide binding activity appears to be conserved in human CDKs.  We have also shown that the subcellular localisation of the Mcm2-7 complex is also regulated by CDKs: CDK activation causes non-chromatin bound Mcm2-7 to be re-localised to the cytoplasm ( 13, 25).

We are also interested in characterising the regulation of pre-RC assembly in human cells ( Niels Mailand).

CDKs inhibit all pre-RC components.