Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei

Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery.     

Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase- like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M- phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine- treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.     

Aphidicolin triggers a block to replication origin firing in Xenopus egg extracts

DNA replication origins are located at random with respect to DNA sequence in Xenopus early embryos and on DNA replicated in Xenopus egg extracts. We have recently shown that origins fire throughout the S phase in Xenopus egg extracts. To study the temporal regulation of origin firing, we have analyzed origin activation in sperm nuclei treated with the DNA polymerase inhibitor aphidicolin. Sperm chromatin was incubated in Xenopus egg extracts in the presence of aphidicolin and transferred to a fresh extract, and digoxigenin-dUTP and biotin-dUTP were added at various times after aphidicolin release to selectively label early and late replicating DNA. Molecular combing analysis of single DNA fibers showed that only a fraction of potential origins were able to initiate in the presence of aphidicolin. After release from aphidicolin, the remaining origins fired asynchronously throughout the S phase. Therefore, initiation during the S phase depends on the normal progression of replication forks assembled at earlier activated origins. Caffeine, an inhibitor of the checkpoint kinases ATR and ATM, did not relieve the aphidicolin-induced block to origin firing. We conclude that a caffeine-insensitive intra-S phase checkpoint regulates origin activation when DNA synthesis is inhibited in Xenopus egg extracts.     

DNA replication reaction in Xenopus cell-free system is suppressed by high pressure

Previously, we demonstrated that when mouse erythroleukemia cells are exposed to a pressure of 80 MPa, the cell-cycle progression of S-phase cells is retarded. To examine the effects of high pressure on DNA replication, we used a Xenopus cell-free system. From cell-cycle progression of sperm nuclei, it was found that sperm nuclei are stable to a pressure of 80 MPa, whereas egg extracts are susceptible to high pressure. Similarly, biotin-16-dUTP was incorporated into 80 MPa-treated sperm nuclei in pressure-untreated extracts, but not into naive sperm nuclei in 80 MPa-treated extracts. These results indicate that DNA replication in Xenopus cell-free system is suppressed by the susceptibility of the extracts to a pressure of 80 MPa.     

Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication.     

p97 ATPase, an ATPase involved in membrane fusion, interacts with DNA unwinding factor (DUF) that functions in DNA replication

DNA unwinding factor (DUF) unwinds duplex DNA and is supposed to function in DNA replication in Xenopus egg extracts. Here we report the isolation and analysis of a DUF-interacting factor. By immunoprecipitation, we found that p97 ATPase (p97) interacts with DUF in Xenopus egg extracts. This interaction was confirmed by the in vitro binding of purified p97 with DUF. When sperm chromatin was added to Xenopus egg extracts to construct nuclei active in DNA replication, p97 was incorporated into the nuclei. These data suggest that the complex of DUF and p97 may function in DNA replication.     

Replication origins in Xenopus egg extract Are 5-15 kilobases apart and are activated in clusters that fire at different times

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [(3)H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5-15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5-10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.     

Replication of Xenopus erythrocyte nuclei in a homologous egg extract requires prior proteolytic treatment

Reactivation and reinitiation of DNA replication in quiescent frog erythrocyte nuclei has been analyzed following incubation in extracts prepared from activated Xenopus eggs. Nuclear decondensation and DNA synthesis only occurred if nuclei were pretreated with low doses of trypsin. This protease treatment did not digest histones, but did degrade several nonhistone proteins. Activated erythrocyte nuclei swell and begin DNA synthesis by 30 min after being mixed with the egg extract. In some extracts virtually complete genome replication was achieved in all nuclei after 2-3 hr. Addition of several protease inhibitors during sperm nuclear isolation significantly reduced the template efficiency of these preparations. We concluded that proteolytic alteration of nonhistone nuclear structural proteins may be a general mechanism which permits quiescent nuclei to reenter the replication cycle. Erythrocyte nuclei and egg extracts provide an excellent experimental system in which to investigate the processes of nuclear reactivation.     

A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs

BACKGROUND: Alteration of chromatin structure is a key step in various aspects of DNA metabolism. DNA unwinding factors such as the high mobility group (HMG) proteins are thought to play a general role in controlling chromatin structure and a specific role in controlling DNA replication. For instance, in the in vitro simian virus 40 replication system, minichromosomes containing HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unfolding the higher-order chromatin structure. At present, however, only limited data suggest an involvement of DNA unwinding factors in DNA replication. RESULTS: We purified from Xenopus eggs a novel heterodimeric factor, termed DNA unwinding factor (DUF), that consists of 87 kDa and 140 kDa polypeptides. DUF unwinds closed-circular duplex DNA in the presence of topoisomerase I, but it does not possess a DNA gyrase activity: it does not introduce negative supercoils into DNA at the expense of ATP hydrolysis. Cloning and sequencing of the cDNAs encoding the two polypeptides revealed that the 87 kDa polypeptide is homologous to a mammalian HMG protein, T160/structure-specific recognition protein. The 140 kDa polypeptide is homologous to yeast Cdc68, a protein that controls the expression of several genes during the G1 phase of the cell cycle by modulating chromatin structure. Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the extract to replicate exogenously added sperm chromatin or plasmid DNA. CONCLUSIONS: We propose that DUF plays a role in DNA replication in Xenopus egg extracts.     

Expression of ISWI and its binding to chromatin during the cell cycle and early development

We have characterized Xenopus ISWI, a catalytic subunit of a family of chromatin-remodeling complexes. We show that ISWI is expressed constitutively during development but poorly expressed in adult tissues except oocytes which contain a large store of maternal protein. We further analyzed its localization both in vivo and in vitro in Xenopus cell cycle extracts and identified that ISWI binds to chromatin at the G1-S period. However, its association to chromatin does not require ongoing DNA replication. Immunodepletion of ISWI has no effect on either sperm chromatin decondensation or the kinetics and efficiency of DNA replication. Nucleosome assembly also occurs in ISWI-depleted extracts, but nucleosome spacing is disturbed. From these results, we conclude that ISWI is not necessary for sperm chromatin decondensation and the accelerated rates of DNA replication that characterize early development.     

Analyzing the ATR-mediated checkpoint using Xenopus egg extracts

Our knowledge of cell cycle events such as DNA replication and mitosis has been advanced significantly through the use of Xenopus egg extracts as a model system. More recently, Xenopus extracts have been used to investigate the cellular mechanisms that ensure accurate and complete duplication of the genome, processes otherwise known as the DNA damage and replication checkpoints. Here we describe several Xenopus extract methods that have advanced the study of the ATR-mediated checkpoints. These include a protocol for the preparation of nucleoplasmic extract (NPE), which is a soluble extract system useful for studying nuclear events such as DNA replication and checkpoints. In addition, we describe several key assays for studying checkpoint activation as well as methods for using small DNA structures to activate ATR.     

A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation.

The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport. RNA processing and cell cycle checkpoint control. A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran. We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes. In these studies, we employed mutant Ran proteins to perturb nuclear assembly and function. The addition of a bacterially expressed mutant form of Ran (T24N-Ran), which was predicted to be primarily in the GDP-bound state, profoundly disrupted nuclear assembly and DNA replication in extracts. We further examined the molecular mechanism by which T24N-Ran disrupts normal nuclear activity and found that T24N-Ran binds tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N-Ran-blocked interphase extracts to assemble nuclei from de-membranated sperm chromatin and to replicate their DNA could be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity. Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high levels of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts.     

DNA replication in cell-free extracts from Drosophila melanogaster.

We have developed an efficient in vitro replication system from 0-2 h Drosophila melanogaster embryos. Demembranated Xenopus sperm DNA when incubated in such an extract first becomes enclosed in a nucleus-like structure with a nuclear envelope and a karyoskeleton. It then undergoes one round of semiconservative replication--this replication appears completely dependent on nuclear formation. Up to 30% of input DNA is nucleated in one reaction. Efficient nuclear formation and replication are dependent on a cold treatment step, prior to disruption of the embryos. They also depend on the age of the embryos used. Extracts from older embryos (0-5 h) are capable of nuclear formation, although at a much reduced efficiency, and repair synthesis, but seem to have lost the ability to initiate DNA replication. In addition to replicating sperm DNA this system appears capable of carrying out semi-conservative replication on some plasmids. However, it cannot use these to trigger nuclear formation; replication is only seen if the plasmids are coincubated with sperm DNA. The in vitro formed nuclei have not been observed to trigger nuclear envelope breakdown and entry into mitosis. However, they can re-replicate the DNA if the nuclei are permeabilized. This system should be a useful complement to the previously isolated Xenopus in vitro replication system. In addition the amenability of Drosophila to genetic study should open up new approaches not previously possible with Xenopus.     

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.     

Periodic DNA synthesis in cell-free extracts of Xenopus eggs.

Cell-free extracts prepared from unfertilized eggs of Xenopus laevis support DNA synthesis on sperm pronuclei. Continuous labelling studies using [3H]dCTP and pulse labelling studies using [32P]dCTP demonstrate that synthesis occurs in short bursts of 40 min, which are punctuated by periods of 20-40 min during which no synthesis occurs. Density substitution experiments using bromodeoxyuridine demonstrate that this synthesis involves the initiation of replication and reveals that re-initiation events can occur following multiple bursts of replication. The periodic properties of these extracts are sensitive to protein synthesis inhibitors.     

Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis.

The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.     

Control of the Cdc2/cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors.

Proliferating eukaryotic cells possess checkpoint mechanisms that block cell division in the presence of unreplicated or damaged DNA. Using cell-free extracts from Xenopus eggs, we have investigated the mechanisms underlying the inability of a recombinant Cdc2/cyclin B complex to induce mitosis in the presence of incompletely replicated DNA. We found that the activities of the kinases and phosphatases that regulate the major phosphorylation sites on Cdc2 (e.g., tyrosine 15, threonine 14, and threonine 161) are not altered significantly under conditions where Xenopus extracts remain stably arrested in interphase due to the presence of the replication inhibitor aphidicolin. However, at threshold concentrations, a Cdc2/cyclin B complex containing a mutant Cdc2 subunit that cannot be phosphorylated on either tyrosine 15 or threonine 14 displays a markedly reduced capacity to induce mitosis in the presence of aphidicolin. This observation indicates that the replication checkpoint in Xenopus egg extracts functions without the inhibitory tyrosine and threonine phosphorylation of Cdc2. We provide evidence that the checkpoint-dependent suppression of the Cdc2/cyclin B complex involves a titratable inhibitor that is regulated by the presence of unreplicated DNA.     

Partial purification of Xenopus laevis egg extract factor(s) that induce swelling in permeabilized human sperm

A combination of adsorption (protamine-agarose) and gel filtration (Sephacryl S-300) chromatography was used to enrich for factor(s) in Xenopus laevis frog egg extract that induce nuclear swelling-chromatin decondensation in permeabilized human sperm. It was determined that a 70-fold purification of the factor(s) that induce human sperm nuclear swelling has resulted from the purification scheme. The reduced, active factor(s) have a Kav of 0.12 and an approximate molecular weight of 290,000 daltons. The extract nuclear swelling activity is sensitive to temperatures of 50 degrees C and above, as well as proteolytic treatment. RNase and cycloheximide treatments of the extracts have no effect on the nuclear swelling activity. These data suggest that the egg extract factor(s) that induce nuclear swelling in permeabilized human sperm are protein(s) that are present in the unfertilized frog egg.