John W. Newport

The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis

In Xenopus, a cytoplasmic agent known as MPF induces entry into mitosis. In fission yeast, genetic studies have shown that the cdc2 kinase regulates mitotic initiation. The 13 kd product of the suc1 gene interacts with the cdc2 kinase in yeast cells. We show that the yeast suc1 gene product (p13) is a potent inhibitor of MPF in cell-free extracts from Xenopus eggs. p13 appears to exert its antagonistic effect by binding directly to MPF. MPF activity is quantitatively depleted by chromatography on a p13 affinity column. Concomitantly, the Xenopus counterpart of the yeast cdc2 protein is adsorbed to the column. A 42 kd protein also binds specifically to the p13 affinity matrix. These findings suggest that the Xenopus cdc2 protein and the 42 kd protein are components of MPF.

Nuclear reconstitution in vitro: Stages of assembly around protein-free DNA

We have developed a cell-free system derived from Xenopus eggs that reconstitutes nuclear structure around an added protein-free substrate (bacteriophage lambda DNA). Assembled nuclei are morphologically indistinguishable from normal eukaryotic nuclei: they are surrounded by a double membrane containing nuclear pores and are lined with a peripheral nuclear lamina. Nuclear assembly involves discrete intermediate steps, including nucleosome assembly, scaffold assembly, and nuclear membrane and lamina assembly, indicating that during reconstitution nuclear organization is assembled one level at a time. Topoisomerase II inhibitors block nuclear assembly. Lamin proteins and membrane vesicles bind to chromatin late in assembly, suggesting that these components do not interact with chromatin that is formed early in assembly. Reconstituted nuclei replicate their DNA; replication begins only after envelope formation has initiated, indicating that envelope attachment may be important for regulating replication.

Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes

Xenopus eggs contain two distinct cdc2 homologs of 34 and 32 kd. We show that the 32 kd cdc2 protein, like the 34 kd protein, is a kinase. However, unlike the 34 kd homolog, the 32 kd cdc2 kinase activity does not decrease dramatically at the end of mitosis. The 32 kd protein does not associate with mitotic cyclins B1 and B2 but does associate with cyclin A and a novel doublet of proteins of 54 kd that may regulate its activity. We also show that depletion of the 32 kd cdc2 homolog from a Xenopus extract blocks DNA replication, but does not inhibit entry into mitosis. By contrast, depletion of the 34 kd cdc2 homolog or absence of mitotic cyclins from an extract does not inhibit replication, but does block entry into mitosis. Our results indicate that in higher eukaryotes, DNA replication (G1-S) and mitosis (G2-M) may be controlled by distinctly different cdc2 proteins.

Initiation of Eukaryotic DNA Replication: Origin Unwinding and Sequential Chromatin Association of Cdc45, RPA, and DNA Polymerase α

We report that a plasmid replicating in Xenopus egg extracts becomes negatively supercoiled during replication initiation. Supercoiling requires the initiation factor Cdc45, as well as the single-stranded DNA-binding protein RPA, and therefore likely represents origin unwinding. When unwinding is prevented, Cdc45 binds to chromatin whereas DNA polymerase alpha does not, indicating that Cdc45, RPA, and DNA polymerase alpha bind chromatin sequentially at the G1/S transition. Whereas the extent of origin unwinding is normally limited, it increases dramatically when DNA polymerase alpha is inhibited, indicating that the helicase that unwinds DNA during initiation can become uncoupled from the replication fork. We discuss the implications of these results for the location of replication start sites relative to the prereplication complex.

Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: Studies in Xenopus

During cell division complete DNA replication must occur before mitosis is initiated. Using a cell-free extract derived from Xenopus eggs that oscillates between S phase and mitosis, we have investigated how completion of DNA synthesis is coupled to the initiation of mitosis. We find that Xenopus eggs contain a feedback pathway which suppresses mitosis until replication is completed and that activation of this inhibitory system is dependent on the presence of a threshold concentration of unreplicated DNA. We demonstrate that in the presence of unreplicated DNA the active feedback system inhibits initiation of mitosis by blocking the activation of MPF, a regulator of mitosis found in all eukaryotic cells. Our results demonstrate that the feedback system does not inhibit MPF activation by blocking the synthesis or accumulation of cyclin protein, a subunit of MPF, or by blocking association of cyclin with the cdc2 subunit of MPF. We propose that the feedback system blocks mitosis by maintaining MPF in an inactive state by modulating posttranslational modifications critical for MPF activation.

Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes.

We describe a stable cell-free mitotic extract derived from Xenopus eggs that contains activities necessary for nuclear envelope breakdown and chromosome condensation during mitosis. Using these cell-free extracts, we have demonstrated that nuclear envelope vesicularization, lamina solubilization, and chromosome condensation are independent and separable biochemical processes. We present evidence indicating that during mitosis nuclear membrane breakdown may involve the binding of a coating protein, lamin solubilization is enzymatically driven, and chromosome condensation involves both binding proteins and enzymatic activities including topoisomerase II. These results provide a coherent framework for investigating structural modification of the nucleus during mitosis at the biochemical level.

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids. I. Characterization of the binding interactions.

Abstract In this paper we examine molecular details of the interaction of bacteriophage T4-coded gene 32 protein with oligo- and polynucleotides. It is shown that the binding affinity ( K oligo ) of oligonucleotides of length ( l ) from two to eight nucleotide residues for gene 32 protein is essentially independent of base composition or sugar type. This binding also shows little dependence on salt concentration and on oligonucleotide length; even the expected statistical length factor in K oligo is not observed, suggesting that binding occurs at the end of the oligonucleotide lattice and that the oligonucleotide is not free to move across the binding site. Co-operative (contiguous) or isolated binding of gene 32 protein to polynucleotides is very different; here binding is highly salt dependent ( ∂ log Kω ∂ log [NaCl] ∼- −7 ) and essentially stoichiometric at salt concentrations less than ~0.2 m (for poly(rA)). Binding becomes much weaker and the binding isotherms appear typically co-operative (sigmoid) in protein concentration at higher salt concentrations. We demonstrate, by fitting the co-operative binding isotherms to theoretical plots at various salt concentrations and also by measuring binding at very low protein binding density (ν), that the entire salt dependence of Kω is in the intrinsic binding constant ( K ); the co-operativity parameter (ω) is essentially independent of salt concentration. Furthermore, by determining titration curves in the presence of salts containing a series of different anions and cations, it is shown that the major part of the salt dependence of the gene 32 protein-polynucleotide interaction is due to anion (rather than to cation) displacement effects. Binding parameters of oligonucleotides of length sufficient to bind two or more gene 32 protein monomers show behavior intermediate between the oligonucleotide and the polynucleotide binding modes. These different binding modes probably reflect different conformations of the protein; the results are analyzed to produce a preliminary molecular model of the interactions of gene 32 protein with nucleic acids in its different binding modes.

Regulated Chromosomal DNA Replication in the Absence of a Nucleus

Using Xenopus egg extracts, we have developed a completely soluble system for eukaryotic chromosomal DNA replication. In the absence of a nuclear envelope, a single, complete round of ORC-dependent DNA replication is catalyzed by cytosolic and nuclear extracts added sequentially to demembranated sperm chromatin or prokaryotic plasmid DNA. The absence of rereplication is explained by an activity present in the nucleus that prevents the binding of MCM to chromatin. Our results indicate that the role of the nuclear envelope in DNA replication is to concentrate activators and inhibitors of replication inside the nucleus. In addition, they provide direct evidence that metazoans use the same strategy as yeast to activate DNA replication and to restrict it to a single round per cell cycle.

Cdk2 Kinase Is Required for Entry into Mitosis as a Positive Regulator of Cdc2–Cyclin B Kinase Activity

In higher eukaryotes, Cdk2 kinase plays an essential role in regulating the G1-S transition. Here, we use cycling Xenopus egg extracts to examine the requirement of Cdk2 kinase on progression into mitosis. Interestingly, when Cdk2 kinase activity is inhibited by the Cdk-specific inhibitor, p21Cip1, a block to mitosis occurs, and inactive Cdc2-cyclin B accumulates. This block occurs in the absence of nuclei and is not due to direct inhibition of Cdc2 by Cip. Importantly, this block to mitosis is reversible by restoring Cdk2-cyclin E kinase activity to a Cip-treated cycling extract. Moreover, immunodepletion of Cdk2 from interphase extracts prevents activation of Cdc2 upon the addition of exogenous cyclin B. Thus, our data show that Cdk2 kinase is a positive regulator of Cdc2-cyclin B complexes and establish a link between Cdk2 kinase and cell cycle progression into mitosis.

Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2

In cell-free extracts derived from Xenopus eggs which oscillate between S phase and mitosis, incompletely replicated DNA blocks the activation of p34cdc2-cyclin by maintaining p34cdc2 in a tyrosine-phosphorylated form. We used a recombinant cyclin fusion protein to generate a substrate to measure the ability of the tyrosine kinase(s) to phosphorylate and inactivate p34cdc2 in the absence of tyrosine phosphatase activity. p34cdc2 tyrosine phosphorylation is highly regulated during the cell cycle, being elevated in S phase and attenuated in mitosis. The elevation in p34cdc2 tyrosine phosphorylation rate occurs in response to the presence of incompletely replicated DNA. Moreover, okadaic acid and caffeine, which uncouple the dependence of mitosis on the completion of S phase, increase unphosphorylated p34cdc2 by attenuating tyrosine kinase function. These data indicate that the control system, which monitors the state of DNA replication, modulates the function of the tyrosine kinase by a phosphorylation/dephosphorylation mechanism, ensuring that mitosis occurs only when S phase is complete.