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To prevent re-replication of DNA, the licensing of replication origins is inhibited in S phase and G2. New work shows that the small GTPase Ran can directly inhibit licensing inside nuclei once CDKs are active late in the cell cycle.
The small GTPase Ran plays a central role in a number of nuclear processes including nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation (Weis, 2003). Ran’s GTP exchange factor, RCC1, is bound to chromatin, whilst its GTPase-activating protein, RanGAP, is located in the cytoplasm. This creates a steep Ran gradient, with the GTP-bound form predominating in the nucleus (or close to the chromosomes in mitosis) and the GDP-bound form predominating in the cytoplasm. In this issue, Ryuji Yamaguchi and John Newport describe a new function for Ran that helps ensure the precise duplication of chromosomal DNA in each cell cycle. This surprising addition to Ran’s repertoire again depends on its ability to distinguish the nucleoplasm from the cytoplasm.
The replication of eukaryotic chromosomal DNA requires the initiation of replication forks from thousands of replication origins. These must be regulated so that none fires more than once in each cell cycle. The cell achieves this by breaking the initiation process into two non-overlapping phases. In the first phase, occurring in late mitosis and early G1, replication origins are “licensed” for replication by assembly of a pre-replicative complex (pre-RC) of initiation proteins. When replication forks are initiated at licensed replication origins during the subsequent S phase, the pre-RC is disassembled, converting the origin to the unlicensed state incapable of supporting further initiation. In order for this system to work properly, the licensing system that assembles new pre-RCs must shut down before S phase starts. Yamaguchi and Newport now show that Ran plays an unexpectedly direct role in this shut-down.
Previous work had shown that inactivation of the licensing system prior to S phase involves the separation of the inside of the nucleus from the outside. Using extracts of Xenopus eggs that support cell cycle progression in vitro, it was found that in order for replicated G2 nuclei to become competent for a further round of DNA replication their nuclear envelope had to be transiently permeabilized, an event that normally occurs during mitosis. This suggested that some essential initiation factor – the licensing factor – was physically excluded by the nuclear envelope (Blow and Hodgson, 2002 and references therein). Subsequent work has shown that several pre-RC components are indeed excluded from the nucleus during S and G2 phases. In metazoans the soluble pool of the pre-RC component Cdc6 is exported out of the nucleus in late G1 whilst in yeast two other pre-RC components, Cdt1 and Mcm2-7, are exported instead (Blow and Hodgson, 2002; Nishitani and Lygerou, 2002; Tanaka and Diffley, 2002 and references therein).
Yamaguchi and Newport’s starting point was to ask why nucleoplasmic extract (NPE) of Xenopus eggs cannot assemble pre-RCs on added DNA (Walter et al., 1998). They noted that such nuclear extracts would be expected to have high Ran-GTP levels, and examined the effect of adding a mutant form of Ran, RanT24N, which cannot bind GTP. Surprisingly, RanT24N allowed the nucleoplasmic extract to assemble pre-RCs, as did treatment of the extracts with a combination of proteins (RanGAP and RanBP1) expected to destabilize Ran-GTP. Conversely, a Ran mutant unable to hydrolyze GTP, RanQ69L, inhibited licensing when added to the cytoplasm. These results suggest a direct inhibition of the licensing system by Ran-GTP and provide an elegant explanation for why pre-RC assembly cannot take place within intact nuclei in the Xenopus system.
How does Ran-GTP inhibit licensing? Ran’s key function in nucleocytoplasmic transport is to drive the assembly and disassembly of complexes between nuclear import/export receptors and their cargo. The export receptor Crm1 can only bind its cargo when bound to Ran-GTP. Once it enters the cytoplasm, RanGAP converts the Ran-GTP in the export complex to Ran-GDP and the complex dissociates. Yamaguchi and Newport show that in both nucleoplasmic and cytoplasmic extracts Crm1 can form a complex with the Mcm2-7 component of the pre-RC (Figure 1). The quantity of this complex is increased by RanQ69L and is decreased by RanT24N, suggesting that Mcm2-7 binding to Crm1 follows similar rules to that of other export complexes. Evidence that the inhibition of licensing by Ran-GTP involves Crm1 comes from the observation that the ability of RanQ69L to inhibit licensing in cytoplasmic extracts depends on the presence of Crm1. However, no direct evidence is presented that Mcm2-7 bound to Crm1 is inactive for licensing, and it remains possible that there are other targets. For example, the licensing inhibitor geminin is activated following nuclear import in the Xenopus system, and this activation is known to require Ran function (Hodgson et al., 2002).
In normal somatic cell cycles, pre-RC assembly can take place during G1 phase, even though the DNA is inside an intact nucleus with high levels of Ran-GTP. At face value this seems to be at odds with the inhibitory effect of Ran-GTP described by Yamaguchi and Newport. There is, however, an additional factor to be considered – the role of cyclin-dependent kinases (CDKs), which drive progression through all the major events of the cell division cycle. Experiments originally performed in yeast showed that CDKs directly inhibit the licensing system. This means that in normal cell cycles pre-RCs only form in late mitosis and early G1 when CDK activity is low (Blow and Hodgson, 2002; Nishitani and Lygerou, 2002). In the rapid cell cycles of the early Xenopus embryo there is no distinct G1 period when nuclei have fully assembled but CDKs are inactive. Yamaguchi and Newport show that the ability of RanQ69L to inhibit licensing also depends on CDK activity, consistent with the idea that inhibition normally only occurs inside nuclei after CDKs have been activated.
Is this the answer to how re-replication of DNA is prevented in a single cell cycle? Although Yamaguchi and Newport show that interfering with the Ran system can drive re-replication, it is unlikely that direct inhibition of licensing by Ran-GTP is the only control. Previous work suggests that several redundant mechanisms might exist to minimize the risk of re-replication occurring, an event with potentially catastrophic consequences. As mentioned above, pre-RC components are known to be exported from the nucleus from late G1 onwards, whilst at the same time geminin becomes activated (interestingly these are both Ran-dependent functions). Several pre-RC components are also degraded at the G1/S phase transition, whilst in yeast it appears likely that licensing is inhibited by direct phosphorylation of some pre-RC components by CDKs (Jallepalli et al., 1997; Nguyen et al., 2001; Vas et al., 2001; Wuarin et al., 2002). Understanding the interplay between these different regulatory systems in different cell types promises to yield further surprises.