DNA replication is a necessary prelude to the division of a eukaryotic cell. Initiation of this process requires a complex script, involving many proteins: details of one of the main acts now emerge.
In 1992, a report by Li and Alberts1 appeared in these pages to comment on a seminal discovery2 — the identification of the protein factors from the budding yeast Saccharomyces cerevisiae that recognize the spots on DNA for initiating the DNA-replication phase of the cell cycle. Finding this 'origin-recognition complex' was one task, however; identifying the participants in the actual switch that starts DNA synthesis is another. This latter achievement is now described in papers by Zegerman and Diffley3, and by Tanaka et al.4, on pages 281 and 328 of this issue. At the heart of this intricate process is the phosphorylation — and thus activation — of two proteins known as Sld3 and Sld2, and the involvement of a third named Dpb11.
The pathway to DNA replication begins early in the cell cycle, with events occurring at the end of nuclear division and in the ensuing G1 phase. DNA synthesis — during which a complete single copy of the entire genome is made — then commences in S phase. Transitions between all cell-cycle phases are controlled by the activation and deactivation of a series of cyclin-dependent kinases (CDKs), which control the phosphorylation of other proteins. Thus, after the origin-recognition complex had been identified, finding the actual targets for S-CDK, the CDK known to promote the switch from G1 to S phase, became a major objective.
Understanding the switch required investigations of the complex pathway that brings the almost completed replication machinery to the correct position on the DNA. What emerged first was an understanding of the assembly of a pre-replication complex during G1 (Fig. 1). This requires many of the factors that interact with the origin-recognition complex and low levels of CDK. The step of unwinding the DNA strands, which is carried out by a helicase enzyme, must be one of the earliest steps in DNA synthesis, and an inactive form of such an enzyme arrives at the pre-replication complex early in the pathway. The origin-recognition complex and another player, Cdc6, form a machine that recruits other proteins (minichromosome maintenance proteins, or MCMs) to the origin of DNA replication during G1 (ref. 5); the MCMs eventually provide essential helicase activity during S phase. Other studies6 have revealed how components of the pre-replication complex are destroyed by the S-phase-promoting factors, so allowing for the once-and-only-once initiation of DNA synthesis. But the actual switch to start synthesis remained elusive.
Zegerman and Diffley3 and Tanaka et al.4 now reveal the components of that switch — that is, the targets of the S-CDKs — in S. cerevisiae. They also disclose surprising details about what must be accomplished during G1 for DNA synthesis to begin. The switch probably occurs with the activation of the DNA unwinding process that creates the substrates for polymerase, the enzyme that does the actual work of synthesis, and involves Dpb11, Sld3 and Sld2 — or 11–3–2 for short.
Previous work by Araki and colleagues had shown that Sld2 is an essential target for S-CDK, and that there are genetic and biochemical interactions between Sld2 and Dpb11. Crucially, the phosphorylated form of Sld2 binds tightly to particular parts of Dpb11 known as BRCT repeats. When replacing a normal copy of the gene, expression of an engineered SLD2 gene encoding a 'phosphomimetic' residue could maintain function, but cells still required passage through G1 and functioning S-CDK. This finding implied, but did not prove, that there are other targets for S-CDK. Sld3 contains 12 sites for phosphorylation by CDKs, and genetic interactions between DPB11 and SLD3 brought Sld3 into the experimental spotlight. The new experiments of Zegerman and Diffley3 and Tanaka et al.4 show that the phosphorylation of Sld3 on two amino-acid residues, and of Sld2 on a single residue, are all that are required for the switch to operate. Importantly, the two phosphorylated proteins, Sld2 and Sld3, must bind to Dpb11 (Fig. 1), and this docking presumably occurs at sites marked as origins of DNA replication by the origin-recognition complex.
Armed with this knowledge, the two groups3,4 were able to generate S-CDK 'bypass' conditions, in which there was no need for S-CDK to prompt S phase. This allowed them to tease apart the machinery determining entry into S phase. Their approach involved α-factor, a yeast hormone that arrests cells in G1. Together with genetic manipulations, this factor can be used to investigate the consequences for a strain of yeast that can enter S phase without S-CDK activity.
Under conditions in which such activity is not required, we might have expected premature DNA replication and complete bypass of the G1 phase. We learn from Zegerman and Diffley3 that α-factor-arrested cells with the S-CDK bypass system show only weak DNA replication. G1 is a busy time for cells and it turns out that another essential kinase, Cdc7, has a regulatory subunit, Dbf4, that is degraded early on and only reaches critical levels after the G1-specific CDKs have inactivated the protein-degradation machinery. Among the main targets for the Cdc7–Dbf4 kinase in DNA replication are the MCM proteins7(Fig. 1). Zegerman and Diffley report that, with the bypass system in place and adequate expression of Dbf4, DNA replication can be strong in α-factor G1-arrested cells and, as would be expected, is lethal. So, regulation of both Cdc7 and S-CDK activity is required to prevent premature DNA replication in G1 phase, providing the sequence of events shown in Figure 1. Thus we have a bare skeleton of what must be accomplished during G1 to provide the necessary and sufficient conditions for passage into the next phase of the cell cycle — at least for DNA replication.
What is now needed is a mechanistic insight into what the 11–3–2 complex actually does to initiate DNA replication; whatever that function is, it is transient and not required beyond the step that instigates DNA synthesis8. Some suggestions are detailed in Box 1. This topic will now be the subject of considerable attention.
Meanwhile, I can echo a comment made 15 years ago. In their News & Views article1, Li and Alberts equated the successful identification of the origin-recognition complex to the finding of the holy grail. For cell and molecular biologists, that sentiment can be echoed to express the significance of the discoveries made by Zegerman and Diffley3, and Tanaka and colleagues4.
Li, J. J. & Alberts, B. Nature 357, 114–115 (1992).
Bell, S. P. & Stillman, B. Nature 357, 128–134 (1992).
Zegerman, P. & Diffley, J. F. X. Nature 445, 281–285 (2007).
Tanaka, S. et al. Nature 445, 328–332 (2007).
Randell, J. C. W., Bowers, J. L., Rodriguez, H. K. & Bell, S. P. Mol. Cell 21, 29–39 (2006).
Blow, J. J. & Dutta, A. Nature Rev. Mol. Cell Biol. 6, 476–486 (2005).
Sheu, Y. J. & Stillman, B. Mol. Cell 24, 101–113 (2006).
Kanemaki, M. & Labib, K. EMBO J. 25, 1753–1763 (2006).
Moyer, S. E., Lewis, P. W. & Botchan, M. R. Proc. Natl Acad. Sci. USA 103, 10236–10241 (2006).
Pacek, M. et al. Mol. Cell 21, 581–587 (2006).
Gambus, A. et al. Nature Cell Biol. 8, 358–366 (2006).
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