How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?

Abstract

Chromosome replication occurs precisely once during the cell cycle of almost all eukaryotic cells, and is a highly complex process that is still understood relatively poorly. Two conserved kinases called Cdc7 (cell division cycle 7) and cyclin-dependent kinase (CDK) are required to establish replication forks during the initiation of chromosome replication, and a key feature of this process is the activation of the replicative DNA helicase in situ at each origin of DNA replication. A series of recent studies has shed new light on the targets of Cdc7 and CDK, indicating that chromosome replication probably initiates by a fundamentally similar mechanism in all eukaryotes.

Figure 1.

The initiation of chromosome replication in budding yeast. The MCM2–7 helicase is recruited to origins during the G1 phase of the cell cycle, and is loaded around dsDNA by the Cdc6 and Cdt1 proteins together with the origin recognition complex. Studies of the loading reaction in vitro indicate that the MCM2–7 helicase is loaded as a double hexamer, in contrast to the single-hexameric form in solution, and multiple double hexamers are probably loaded at each origin. This produces the prereplicative complex at origins, and is equivalent to the step referred to in Xenopus as the “licensing reaction.” At the earliest budding yeast origins, the Sld3 protein is recruited in a weakly bound form together with Cdc45, perhaps by direct interaction with the MCM2–7 complex. The MCM2–7 helicase remains inactive until cells enter S phase and activate Cdc7 and CDKs. The Cdc7 kinase phosphorylates the N-terminal tails of Mcm2/4/6 and probably induces a structural change in the MCM2–7 complex. It seems likely that CDK also phosphorylates MCM2–7, but the major targets are Sld2 and Sld3, the phosphorylated forms of which appear to be bridged by Dpb11 (the N-terminal pair of BRCT repeats of Dpb11 binds Sld3, whereas the C-terminal pair of BRCT repeats binds Sld2), allowing the recruitment of GINS and DNA polymerase ɛ to origins. This leads to the stable association of CMG together with a variety of other factors (not shown for simplicity) to form the RPC. Activation of the MCM2–7 helicase unwinds the origin and allows the priming of leading and lagging strands by DNA polymerase α. At present, the nature of the active CMG helicase within the RPC is unclear, as discussed in the text. DNA polymerase ɛ extends the leading strand at each DNA replication fork, and DNA polymerase δ extends each Okazaki fragment on the lagging strand. Many other factors also act at DNA replication forks, but these have been omitted, as they remain beyond the scope of this review. Similar events are thought to occur subsequently at later origins, to which the various initiation factors including Sld3 and Sld2 are recruited only around the time that initiation occurs at each particular origin. At present it is not clear whether CDK phosphorylates Sld3 only in situ at origins, or whether CDK can also phosphorylate Sld3 away from origins.