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Replication licensing factor (RLF) is involved in preventing re-replication of chromosomal DNA in a single cell cycle, and previously has been separated into two components termed RLF-M and RLF-B. Here we show that Xenopus RLF-M consists of all six members of the MCM/P1 protein family, XMcm2-XMcm7. The six MCM/P1 polypeptides co-eluted on glycerol gradients and gel filtration as complexes with a mol. wt of approximately 400 kDa. In crude Xenopus extract, all six MCM/P1 polypeptides co-precipitated with anti-XMcm3 antibody, although only XMcm5 quantitatively co-precipitated from purified RLF-M. Further fractionation separated RLF-M into two sub-components, one consisting of XMcms 3 and 5, the other consisting of XMcms 2, 4, 6 and 7. Neither of the sub-components provided RLF-M activity. Finally, we show that all six MCM/P1 proteins bind synchronously to chromatin before the onset of S-phase and are displaced as S-phase proceeds. These results strongly suggest that complexes containing all six MCM/P1 proteins are necessary for replication licensing.

Eukaryotic DNA replication is regulated at least in part by the assembly of initiation proteins onto origins of replication. The origin recognition complex (ORC) is bound to origins throughout most of the cell cycle. Other initiation proteins, such as Cdc6 and the MCM/P1 proteins, are assembled onto ORC-containing chromatin during G1 to define a prereplicative complex. During S phase, these proteins are displaced from chromatin and their reassembly is inhibited by protein-dependent kinases.

Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 microM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 microM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 microM and 14 microM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 microM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.

Publisher Summary This chapter discusses the characterization of the xenopus replication licensing system. In eukaryotes the entire genome is replicated precisely once during each S phase of the cell cycle. Cell-free extracts of Xenopus eggs support chromosomal DNA replication in vitro, apparently under the same cell cycle controls that exist in vivo. When DNA is added to the cell-free system it is assembled into chromatin, and then into structures resembling normal interphase nuclei, processes that is necessary before the initiation of DNA replication can occur. The precise duplication of chromosomal DNA in the Xenopus cell-free system requires many thousands of replication origins to each fire once and only once. To achieve this, two distinct signals are required for initiation to occur at any given replication origin.

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of "univalents" linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process.

Summary In order for cells to divide, they must replicate their chromosomes precisely once. This replication is performed by a number of proteins that act together to form a replication fork. The major control over DNA replication occurs at replication origins, the sites where active replication forks are initiated. In the yeast S. cerevisiae, origins have been identified as ARSs which possess a defined DNA sequence. These ARSs are recognized by specific proteins including ORC and ABF1. The elements which define origins in higher eukaryotes have not yet been identified. The temporal sequence in which origins fire is dependent on both the chromosomal location of the origin and the transcriptional activity of the dividing cell, with euchromatin and actively-expressed genes tending to replicate early in S-phase. DNA replication is an all-or-nothing event and occurs only after commitment to cell division. Cell fusion studies imply that S-phase inducers must be supplied to a nucleus as a signal to start replication. In addition, cells which have replicated their DNA but not yet undergone mitosis, will not perform further replication, even in the presence of these S-phase inducers. This is currently best explained by the action of a licensing factor, an origin-binding protein which cannot cross the nuclear envelope and so requires a transient nuclear permeabilization in order to make the DNA replication competent. Cyclin-dependent kinases provide good candidates for the S-phase inducers, as cell-cycle regulated activities which can potentially act upon these origin complexes and actually induce initiation to take place. Precise identification of the proteins involved is still to be made.

DNA Replication Licensing Factor (RLF) is an essential activity required to restrict the duplication of genomic DNA to precisely once per cell cycle. Recent fractionation of RLF activity from Xenopus egg extracts has resulted in the identification of two essential components, RLF-B and RLF-M. RLF-M has been purified to homogeneity and has been shown to consist of a complex of proteins in the MCM/P1 family. RLF-B is still unidentified, but possible candidates for this activity have been identified in yeast. Elucidation of the RLF mechanism will provide important insights into the way that chromosome replication is controlled.

Background: Cip1 is a 21 kD protein that interacts with and inhibits cyclin-dependent kinases (cdks). Expression of Cip1 is induced by the tumour suppressor p53, and tumour cells have greatly reduced levels of Cip1. As cdks are required for normal progression through the cell cycle, their inhibition by Cip1 may mediate the ability of p53 to block cell proliferation. Cip1 has also been shown to inhibit the DNA polymerase δ auxiliary factor PCNA (proliferating cell nuclear antigen), which is required for replication-fork elongation, and this could be an alternative mechanism by which p53-induced Cip1 blocks cell proliferation. Results We have investigated the effect of Cip1 protein on chromosomal DNA replication, using cell-free extracts of Xenopus eggs that initiate and complete chromosome replication under normal cell-cycle control. Cip1 protein strongly inhibited an early stage of DNA replication in this system, and this inhibition was not complemented by extracts that had been affinity-depleted of cdks. In contrast, Cip1 did not inhibit the elongation of replication forks that had accumulated in the presence of aphidicolin. Cip1 inhibition of DNA replication was fully rescued by addition of cyclins A or E, but not cyclin B, cdk2 or PCNA. Conclusion Our results suggest that Cip1 specifically blocks the initiation of DNA replication by inhibition of a cyclin-dependent kinase (cdk2), but has no major effect on the elongation of preassembled replication forks. The ability of cyclin A or cyclin E to rescue the Cip1 inhibition suggests that these cyclins may play a direct role in the initiation of replication in the Xenopus system.

While testing purines related to the non-specific protein kinase inhibitors N6-dimethylaminopurine and N6-(delta 2-isopentenyl)adenine as potential inhibitors of the p34cdc2/cyclin B kinase, we discovered a compound with high specificity, 2-(2-hydroxyethylamino)-6- benzylamino-9-methylpurine (olomoucine). Kinetic analysis of kinase inhibition reveals that olomoucine behaves as a competitive inhibitor for ATP and as a non-competitive inhibitor for histone H1 (linear inhibition for both substrates). The kinase specificity of this inhibition was investigated for 35 highly purified kinases (including p34cdk4/cyclin D1, p40cdk6/cyclin D3, cAMP-dependent and cGMP-dependent kinases, eight protein kinase C isoforms, calmodulin-dependent kinase II, myosin light-chain kinase, mitogen-activated S6 kinase, casein kinase 2, double-stranded RNA-activated protein kinase, AMP-stimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell-cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase (and its starfish homologue p44mpk) are substantially inhibited by olomoucine (IC50 values are 7, 7, 7, 3 and 25 microM, respectively). The cdk4/cyclin D1 and cdk6/cyclin D3 kinases are not significantly sensitive to olomoucine (IC50 values greater than 1 mM and 150 microM, respectively). N6-(delta 2-Isopentenyl)adenine is confirmed as a general kinase inhibitor with IC50 values of 50-100 microM for many kinases. The purine specificity of cyclin-dependent kinase inhibition was investigated: among 81 purine derivatives tested, only C2, N6 and N9-substituted purines exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. An essentially similar sensitivity to this olomoucine family of compounds was observed for the brain-specific cdk5/p35 kinase. Structure/activity relationship studies allow speculation on the interactions of olomoucine and its analogues with the kinase catalytic subunit. Olomoucine inhibits in vitro M-phase-promoting factor activity in metaphase-arrested Xenopus egg extracts, inhibits in vitro DNA synthesis in Xenopus interphase egg extracts and inhibits the licensing factor, an essential replication factor ensuring that DNA is replicated only once in each cell cycle. Olomoucine inhibits the starfish oocyte G2/M transition in vivo. Through its unique selectivity olomoucine provides an anti-mitotic reagent that may preferentially inhibit certain steps of the cell cycle.

At mitosis the nuclear envelope (NE) is disassembled to allow chromosome separation. In telophase it is reassembled as the chromosomes decondense. Cell-free extracts of Xenopus eggs have been used extensively to study assembly of the NE and the nuclear pore complexes (NPCs), providing several models for the steps involved. The NE is a surface structure which in cell-free extracts is easily exposed. It is appropriate, therefore, to use a surface imaging technique to study NE dynamics. Field emission in-lens scanning electron microscopy (FEISEM) provides the opportunity to image surfaces, directly, and to visualise details of structures such as the NPC. Here we show the feasibility and value of FEISEM to study the steps of NE formation. Nuclei have been assembled in vitro and fixed at different time points during assembly, followed by conductive staining, platinum coating, and visualisation by FEISEM. Changes on the nuclear surface with time are shown. Details of the surface of chromatin and the cytoplasmic face of NPC structure are demonstrated without the need to isolate the structures from the nucleus.

When a cell replicates and divides, it must copy its DNA. But the DNA must be copied only once per cell-division cycle, so DNA duplication is tightly controlled. A protein called Cdt1 has now been revealed to be a key part of this control mechanism.

The plasticity of replication origin usage during mitosis is associated with longer-term changes to chromatin loop organization., When DNA replication is slowed down, normally dormant replication origins are activated. Recent work demonstrates that cells adapt by changing the organization of chromatin loops and maintaining the new pattern of origin use in subsequent cell cycles.

Extracts of Xenopus eggs efficiently initiate and complete chromosomal DNA replication in vitro, under normal cell cycle controls. Such extracts can be depleted of Xenopus p34cdc2, either by affinity depletion using the protein p13suc1 or by specific immunodepletion. Depleted extracts are incapable of initiating DNA replication, although they efficiently elongate replication forks initiated in undepleted extracts. Depletion of p34cdc2 does not prevent nuclear assembly, which is required for the initiation of DNA replication in this system. Activity can be restored to depleted extracts by readdition of p13suc1 eluates enriched for p34cdc2. These results demonstrate that p34cdc2, or a very closely related protein, is involved in the initiation of chromosomal DNA replication in the cell cycle of higher eukaryotes.

Background Early in the cell cycle a pre-replicative complex (pre-RC) is assembled at each replication origin. This process involves the sequential assembly of the Origin Recognition Complex (ORC), Cdc6, Cdt1 and the MiniChromosome Maintenance (Mcm2-7) proteins onto chromatin to license the origin for use in the subsequent S phase. Licensed origins must then be activated by S phase-inducing cyclin-dependent kinases (S-CDKs) and the Dbf4/Cdc7 kinase.

Quantitative proteomic analyses have traditionally used two-dimensional gel electrophoresis (2DE) for separation and characterisation of complex protein mixtures. Among the difficulties associated with this approach is the solubilisation of protein mixtures for isoelectric focusing (IEF). To find the optimal formulation of the multi-component IEF rehydration buffer (RB) we applied the Taguchi method, a widely used approach for the robust optimisation of complex industrial processes, to determine optimal concentrations for the detergents, carrier ampholytes and reducing agents in RB for 2DE using commercially supplied immobilised pH gradient (IPG) gel strips. Our optimisation resulted in increased protein solubility, improved resolution and reproducibility of 2D gels, using a wide variety of samples. With the updated protocol we routinely detected approximately 4-fold more polypeptides on samples containing complex protein mixtures resolved on small format 2D gels. In addition the pI and size ranges over which proteins could be resolved was substantially improved. Moreover, with improved sample loading and resolution, analysis of individual spots by immunoblotting and mass spectrometry revealed previously uncharacterised posttranscriptional modifications in a variety of chromatin proteins. While the optimised RB (oRB) is specific to the gels and analysis approach we use, our use of the Taguchi method should be generally applicable to a broad range of electrophoresis and analysis systems.

A report on the 2001 Eukaryotic DNA Replication meeting, Cold Spring Harbor Laboratory, New York, 5-9 September 2001.

Plasma lipases and lipid transfer proteins are involved in the generation and speciation of high density lipoproteins. In this study we have examined the influence of plasma lipases and lipid transfer protein activities on the transfer of free cholesterol (FC) and phospholipids (PL) from lipid emulsion to human, rat and mouse lipoproteins. The effect of the lipases was verified by incubation of labeled (3H-FC,14C-PL) triglyceride rich emulsion with human plasma (control, post-heparin and post-heparin plus lipase inhibitor), rat plasma (control and post-heparin) and by the injection of the labeled lipid emulsion into control and heparinized functionally hepatectomized rats. In vitro, the lipase enriched plasma stimulated significantly the transfer of 14C-PL from emulsion to high density lipoprotein (p

We demonstrate that cell-free extracts prepared from activated eggs of X. laevis by a method similar to that of Lohka and Masui initiate and complete semiconservative DNA replication of sperm nuclei and plasmid DNA. The efficiency of replication is comparable to that in the intact egg. Under optimal conditions 70%-100% of nuclei, and up to 38% of naked DNA molecules replicate completely. Genuine initiation of replication occurs rather than elongation of preformed primers or priming of irreversibly denatured templates. Rereplication of templates is observed under certain conditions. In addition to replicating DNA, these extracts also assemble nucleus-like structures from naked DNA.

Summary This paper reviews our current knowledge of the cyclins based on observations of the oocytes and eggs of sea urchins, clams and frogs. Cyclins are proteins found in all eukaryotes whose special property is rapid destruction at specific stages in the cell cycle. The cyclins fall into three families. A-type cyclins have been found in clams, flies and frogs. B-type cyclins have been found in clams, flies, frogs, sea urchins and fission yeast. A more distantly related family of three genes is found in Saccharomyces cerevisiae. B-type cyclins appear to be required for cells to enter mitosis, and their destruction is thought to be necessary for exit from mitosis. We describe evidence in support of these ideas, and describe various conditions under which cyclin destruction is delayed or deranged. We conclude with a discussion of the relationship between the cyclins and maturation- (or M phase-) promoting factor and some ideas on how the cyclins may work.

SummaryFaithful DNA replication ensures genetic integrity in eukaryotic cells, but it is still obscure how replication is organized in space and time within the nucleus. Using timelapse microscopy, we have developed a new assay to analyze the dynamics of DNA replication both spatially and temporally in individual Saccharomyces cerevisiae cells. This allowed us to visualize replication factories, nuclear foci consisting of replication proteins where the bulk of DNA synthesis occurs. We show that the formation of replication factories is a consequence of DNA replication itself. Our analyses of replication at specific DNA sequences support a long-standing hypothesis that sister replication forks generated from the same origin stay associated with each other within a replication factory while the entire replicon is replicated. This assay system allows replication to be studied at extremely high temporal resolution in individual cells, thereby opening a window into how replication dynamics vary from cell to cell.

Summary Xenopus egg extract supports all the major cell-cycle transitions in vitro. We have used a proteomics approach to identify proteins whose abundance on chromatin changes during the course of an in vitro cell cycle. One of the proteins we identified was ELYS/MEL-28, which has recently been described as the earliest-acting factor known to be required for nuclear pore complex (NPC) assembly [1–4]. ELYS interacts with the Nup107-160 complex and is required for its association with chromatin. ELYS contains an AT-hook domain, which we show binds to chromatin with a high affinity. This domain can compete with full-length ELYS for chromatin association, thereby blocking NPC assembly. This provides evidence that ELYS interacts directly with chromatin and that this interaction is essential for NPC assembly and compartmentalization of chromosomal DNA within the cell. Furthermore, we detected a physical association on chromatin between ELYS and the Mcm2-7 replication-licensing proteins. ELYS chromatin loading, NPC assembly, and nuclear growth were delayed when Mcm2-7 was prevented from loading onto chromatin. Because nuclear assembly is required to shut down licensing prior to entry into S phase, our results suggest a mechanism by which these two early cell-cycle events are coordinated with one another.

Cells respond to DNA damage during S phase by slowing chromosome replication. Recent results have shed light on the mechanism by which this ‘intra-S phase’ checkpoint is implemented.

During late mitosis and early interphase, origins of replication become "licensed" for DNA replication by loading Mcm2-7 complexes [1–5]. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7 [6–9]. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase [10]. Here, we show that recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Since the initiation of replication at licensed origins depends on nuclear assembly [11], our results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle.

In eukaryotes the entire genome is replicated precisely once in each cell cycle. No DNA is re-replicated until passage through mitosis into the next S-phase. We have used a cell-free DNA replication system from Xenopus eggs to determine which mitotic changes permit DNA to re-replicate. The system efficiently replicates sperm chromatin, but no DNA is re-replicated in a single incubation. This letter shows that nuclei replicated in vitro are unable to re-replicate in fresh replication extract until they have passed through mitosis. However, the only mitotic change which is required to permit re-replication is nuclear envelope permeabilization. This suggests a simple model for the control of DNA replication in the cell cycle, whereby an essential replication factor is unable to cross the nuclear envelope but can only gain access to DNA when the nuclear envelope breaks down at mitosis.

Our potential for dissecting the complex processes involved in eukaryotic DNA replication has been dramatically increased with the recent development of cell-free systems that recreate many of these processes in vitro. Initial results from these systems have drawn together work on the cell cycle, the enzymology of replication, and the structure of the nucleus.

Cell-free systems from eggs of the frog Xenopus laevis are able to perform most of the acts of eukaryotic chromosome replication in vitro . This now includes the crucial regulatory step of initiation, which had only been achieved for viral systems previously. Purified DNA or nuclei are able to initiate and complete semiconservation replication in egg extracts in vitro (Blow & Laskey, Cell 47, 557-587 (1986)). Replication does not require specialized DNA sequences either in vitro or in microinjected eggs, but in both systems large templates replicate more efficiently than small templates. In some cases replication can re-initiate, excluding the possibility that replication is primed by preexisting primers in the template preparations. When nuclei are replicated in vitro , only one round of replication is observed in a single incubation resembling the single round of replication observed for purified DNA after micro-injection. The mechanism that prevents re-initiation of replication within a single cell cycle is discussed and certain models are eliminated. Nucleosome assembly from histones and DNA has also been studied in cell-free systems from Xenopus eggs. Fractionation has led to the identification of two acidic proteins called nucleoplasmin and N1, which bind histones and transfer them to DNA. The sequences of both proteins have been determined by cDNA cloning and sequencing. Both proteins are found as complexes with histones in eggs.

In each cell cycle the complex structure of the chromosome must be replicated accurately. In the last few years there have been major advances in understanding eukaryotic chromosome replication. Patterns of replication origins have been mapped accurately in yeast chromosomes. Cellular replication proteins have been identified by fractionating cell extracts that replicate viral DNA templates in vitro. Cell-free systems that initiate eukaryotic DNA replication in vitro have demonstrated the importance of complex nuclear architecture in the control of DNA replication. Although the events of S phase were relatively neglected for many years, knowledge of DNA replication is now advancing rapidly in step with other phases of the cell cycle.

Studies on the initiation of DNA replication in eukaryotes have progressed recently through different approaches that promise to converge. Proteins interacting with the origin recognition complex form a prereplicative complex early in the cell cycle. The regulation of the binding of MCM/P1 proteins to chromatin plays a key role in the replication licensing system which prevents re-replication in a single cell cycle. Cyclin-dependent kinases provide an overall control of the cell cycle by stimulating S-phase entry and possibly by preventing re-establishment of prereplicative complexes in G2 phase.

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Cdc7/Dbf4 is a protein kinase that is required for the initiation of DNA replication in eukaryotes. Recent work has provided new clues to the role that Cdc7/Dbf4 plays in this process. A range of other observations suggest that Cdc7/Dbf4 also plays another, less well characterized, role in checkpoint function and in the maintenance of genomic integrity. In this review we attempt to bring together new information to explain how Cdc7/Dbf4 may perform these two distinct functions.

Summary We have developed a cell-free system from frog eggs that efficiently initiates and completes a single round of semi-conservative replication. 70–100 % of sperm chromatin and up to 40 % of plasmid DNA molecules are completely replicated in vitro. Before DNA is replicated it is assembled into nuclei surrounded by a double unit membrane studded with nuclear pores. Flow cytometry shows that initiation events are co-ordinated within individual nuclei, although different nuclei can start to replicate at different times in the same extract. This demonstrates the importance of nuclear structure in the control of DNA replication in this system. Only a single round of semi-conservative replication occurs in the cell-free system. This mirrors the way that only one round of DNA replication occurs in each cell cycle in vivo. When replicated nuclei are transferred to fresh extract they are unable to undergo another round of replication. However, if the nuclear envelope is permeabilised before nuclei are transferred to fresh extract, the DNA becomes capable of undergoing a further round of semi-conservative replication. These results suggest a simple model for the control of DNA replication within the cell cycle, whereby an essential initiation factor can only gain access to DNA when the nuclear envelope breaks down during mitosis.

We have used a novel approach to investigate the control of initiation of replication of sperm nuclei in a Xenopus cell-free extract. Nascent DNA was labelled with biotin by supplementing the extract with biotin-11-dUTP, and isolated nuclei were then probed with fluorescein-conjugated streptavidin. Flow cytometry was used to measure the biotin content of individual nuclei and their total DNA content. This showed that incorporation of the biotinylated precursor increases linearly with DNA content. Haploid sperm nuclei replicate fully to reach the diploid DNA content over 2-6 h in the extract. Synthesis stops once the diploid DNA content is reached. Different nuclei enter S phase at different times over greater than 1.5 h, although they share the same cytoplasmic environment. Nuclei reach their maximum rates of synthesis soon after entry into S phase and some replicate fully in less than 0.5 h, resembling the rates of replication observed in the intact egg. These results indicate that initiations are coordinated within each nucleus such that the nucleus is the fundamental unit of replication in the cell-free system.

metabolites are the true differentiation-inducing factors, but when he analysed the metabolites none had activity and none antagonized the action of DIF itself. The distribution of DIF remains a paradox. The second complication was described 13,,: Jeff Williams (London), who showed that the pre-stalk population, hitherto considered homogeneous, actually consists of three cell types: one is marked by a gene designated 56, one by a gene designated 63, and one does not express either. Williams described how the three populations might cooperate to form the stalk tube of the fruiting body, and provided preliminary data to suggest that the genes 56 and 63 are activated in response to different concentrations of DIF. This would be an exciting result, reminiscent of the genes X and hunchback in Drosophila. And the fact that Dictyosteliunz also has powerful genetics (mutants that are defective in some aspect of development can be maintained as amoebae) may mean that one can now analyse how a single molecule can specify different cell types via extracellular, rather than Antracellular, signalling. The hypothesis of positional information has variously been described (mostly bv those who have not read the original papers) as obvious, meaningless or untestable. This meeting~ held 20 years after the idea was first proposed, provided some direct evidence that positional information is used during development and that it can be analysed at the molecular level.

Purified DNA undergoes a single round of semiconservative replication when incubated in extracts of Xenopus eggs. These extracts also assemble purified DNA into pseudo-nuclei, structures closely resembling normal interphase nuclei. In this paper we show that although less than 60% of purified DNA is assembled into pseudo-nuclei, DNA replication takes place only within these pseudo-nuclei. Further, when nuclear assembly is prevented, the initiation of replication on purified DNA molecules does not occur. In contrast to previous reports, we show that the initiation of DNA replication occurs only during interphase and not during mitosis, even when very high concentrations of purified DNA are used. These experiments show that nuclear formation is a general requirement for the initiation of DNA replication in this system.

The replication licensing system ensures the precise duplication of chromosomal DNA in each cell cycle. In metazoans, a small protein called geminin plays a central role in negatively regulating licensing late in the cell cycle. Recent work using Xenopus egg extracts shows how geminin activity is downregulated on exit from metaphase in a process that requires mitotic cyclin-dependent kinases (CDKs). Geminin is polyubiquitinated by the Anaphase Promoting Complex, but instead of being proteolysed - the normal fate of polyubiquitinated proteins - much of the geminin is deubiquitinated, leaving it inactive. These results suggest a simple model for how precise chromosome duplication is ensured in the Xenopus model system.

In eukaryotes, the initiation of DNA synthesis requires the assembly of a pre-replicative complex (pre-RC) at origins of replication. This involves the sequential binding of ORC (origin-recognition-complex), Cdc6 and MCM proteins, a process referred to as licensing. After origin firing, the Cdc6 and MCM proteins dissociate from the chromatin, and do not rebind until after the completion of mitosis, thereby restricting replication to a single round in each cell cycle. Although nuclei normally become licensed for replication as they enter G(1), the extent to which the license is retained when cells enter the quiescent state (G(0)) is controversial. Here we show that the replication capacity of nuclei from Swiss 3T3 cells, in Xenopus egg extracts, is not lost abruptly with the onset of quiescence, but instead declines gradually. The decline in replication capacity, which affects both the number of nuclei induced to replicate and their subsequent rate of DNA synthesis, is accompanied by a fall in the level of chromatin-bound MCM2. When quiescent nuclei are incubated in egg extracts, they do not bind further MCMs unless the nuclei are first permeabilized. The residual replication capacity of intact nuclei must therefore be dependent on the remaining endogenous MCMs. Although high levels of Cdk activity are known to block MCM binding, we show that the failure of intact nuclei in egg extracts to increase their bound MCMs is not due to their uptake and accumulation of Cdk complexes. Instead, the failure of binding must be due to exclusion of some other binding factor from the nucleus, or to the presence within nuclei of an inhibitor of binding other than Cdk activity. In contrast to the situation in Xenopus egg extracts, following serum stimulation of intact quiescent cells, the level of bound MCMs does increase before the cells reach S phase, without any disruption of the nuclear envelope.