Replication is limited to a single round per cell cycle, and re-replication in metazoans is prevented by a regulatory mechanism that involves Cdk2 kinase. Ryuji Yamaguchi and John Newport now report in Cell the identification of other components of this Cdk2-dependent mechanism.

Yamaguchi and Newport noticed that, when they added sperm chromatin to Xenopus egg cytosol, pre-replication complexes (pre-RCs) assembled rapidly onto sperm DNA. By contrast, when adding sperm chromatin to nuclear extract, MCM helicase — an initiation factor and component of the pre-RC — failed to bind to the chromatin. As nuclear extract contains a high concentration of Ran–GTP compared with the cytosol, and given its many functions in nuclear processes, the authors examined whether Ran–GTP is responsible for preventing assembly of MCM into pre-RCs. They showed that addition to nuclear extract of a mutant Ran protein, RanT24N, which binds weakly to GDP and not to GTP, rescued MCM assembly into pre-RCs, whereas wild-type Ran did not.

Next, the authors wanted to know whether RanT24N had the same effect in intact nuclei. As expected, MCM binding was inhibited in pre-formed nuclei that were supplemented with cytosol containing MCM, and indeed, this inhibition could be reversed by addition of RanT24N. Moreover, in the presence of RanT24N, replication was faster and continued for longer, and bromodeoxyuridine (BrdU) incorporation confirmed that RanT24N induced multiple rounds of replication. When RanT24N was added to reconstituted nuclei immediately after replication (G2 nuclei), MCM re-associated with the chromatin and replication resumed.

By increasing the low cytosolic concentration of Ran–GTP, binding of MCM to chromatin was reduced. Moreover, in the presence of a Ran mutant, RanQ69L — which can bind but not hydrolyse GTP — MCM loading was completely blocked. Yamaguchi and Newport postulated that the low Ran–GTP concentration in the cytosol allows MCM to assemble into pre-RCs prior to nuclear assembly.

Because the nuclear Ran–GTP level is constantly high, the authors hypothesized that Ran–GTP must link to another regulatory component to allow pre-RC formation during G1. Cdk2 kinase is a likely candidate, as it is known to be essential for inhibiting re-replication. When Cdk2 was inactivated by Cdk2 inhibitor p27Kip1, MCM loading was no longer inhibited by RanQ69L. So, Ran–GTP-induced MCM inhibition is dependent on Cdk2.

Using an anti-MCM7 antibody, Yamaguchi and Newport immunoprecipitated the nuclear-export protein exportin-1/Crm1. Moreover, increasing the Ran–GTP concentration by addition of RanQ69L increased the association of Crm1 with MCM. By contrast, addition of RanT24N destabilized the Crm1–MCM complex. Immunodepletion of Crm1 meant that RanQ69L could no longer inhibit MCM assembly, and inhibition could be restored by adding Crm1 back to the extract.

So, the authors have identified yet another nuclear function for the small GTPase Ran, and unravelled one of possibly several (redundant) mechanisms for preventing re-replication in metazoans.