In vertebrates, different complexes of cyclins and cyclin-dependent kinases (Cdks) trigger S phase and mitosis. In Xenopus egg extracts, Cdk2–cyclin E can promote entry into S phase, but not mitosis — the reverse is true for Cdk1–cyclin B. What is responsible for this specificity? In yeast, a single B-type cyclin can be made to promote entry into both S phase and mitosis. So, can Cdk1–cyclin B be made to support DNA replication in vertebrates?

To answer these questions, Tim Hunt and colleagues removed two possible reasons for the inability of Cdk1–cyclin B to support replication — its switch-like response to increasing cyclin B levels and its predominantly cytoplasmic localization.

Cyclin E only supports replication in the nucleus, and cyclin B1 is kept out of the nucleus by a nuclear export signal (NES). So, the authors created several fusion proteins that they introduced into Xenopus egg extracts in which cyclin E was depleted and replication reduced to 10–15% of control levels. They found that glutathione-S-transferase (GST)–cyclin B1ΔN120 (no NES) did localize, albeit slowly, to the nucleus, but couldn't support replication. By contrast, GST–cyclin E–B1 (no NES, but has the nuclear localization signal (NLS) of cyclin E) not only localized to the nucleus but supported replication too, as did GST–cyclin B1ΔN120 plus the NLS of the SV40 large T antigen. This indicated that an NLS is enough for cyclin B1 to support replication.

Hunt and colleagues then looked at the timing of replication and mitosis in the supplemented extracts, as the concentrations of GST–cyclin E–B1 that restored replication overlapped with those that initiated mitosis. Most replication was restored by 200 nM GST–cyclin E–B1 but a higher/lower concentration was ineffective. At this concentration, replication ceased after ∼1 h in time with entry into mitosis. By adding a high concentration of the catalytic domain of the Cdc25B phosphatase (an activator of the Cdk1–cyclin B kinase activity) to overcome the inhibitory effects of the Wee1 and Myt1 kinases on Cdk1, the authors reduced the concentration of cyclin E–B1 needed to restore replication to 80 nM — a concentration that did not trigger mitosis. Cdc25B also allowed GST–cyclin B1ΔN120 to support replication, but not GST–cyclin B1ΔN82, which retains its NES.

So, Cdk1–cyclin B1 can support replication if it is localized in the nucleus. And the fact that there is a block on such a supporting role suggests “...that access to substrates in time and space plays a critical role in determining the function of particular Cdk–cyclin complexes.”