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.     

Histone H1 Reduces the Frequency of Initiation in Xenopus Egg Extract by Limiting the Assembly of Prereplication Complexes on Sperm Chromatin

Somatic histone H1 reduces both the rate and extent of DNA replication in Xenopus egg extract. We show here that H1 inhibits replication directly by reducing the number of replication forks, but not the rate of fork progression, in Xenopus sperm nuclei. Density substitution experiments demonstrate that those forks that are active in H1 nuclei elongate to form large tracts of fully replicated DNA, indicating that inhibition is due to a reduction in the frequency of initiation and not the rate or extent of elongation. The observation that H1 dramatically reduces the number of replication foci in sperm nuclei supports this view. The establishment of replication competent DNA in egg extract requires the assembly of prereplication complexes (pre-RCs) on sperm chromatin. H1 reduces binding of the pre-RC proteins, XOrc2, XCdc6, and XMcm3, to chromatin. Replication competence can be restored in these nuclei, however, only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in egg extract by preventing the assembly of pre-RCs on sperm chromatin, thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 plays a role in regulating replication origin use during Xenopus development.     

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.     

Assembly of correct kinetochore architecture in Xenopus laevis egg extract requires transition of sperm DNA through interphase

Xenopus egg extracts provide a powerful tool for studying formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one employs direct assembly of chromatin from sperm nuclei in CSF-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step, followed by re-establishing of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: the amounts of outer kinetochore proteins such as Bub1, BubR1 and Dynactin subunit p150glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. In contrast, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results argue that transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.     

The sperm nuclear matrix is required for paternal DNA replication

The mammalian sperm nucleus provides an excellent model for studying the relationship between the formation of nuclear structure and the initiation of DNA replication. We previously demonstrated that mammalian sperm nuclei contain a nuclear matrix that organizes the DNA into loop domains in a manner similar to that of somatic cells. In this study, we tested the minimal components of the sperm nucleus that are necessary for the formation of the male pronucleus and for the initiation of DNA synthesis. We extracted mouse sperm nuclei with high salt and dithiothreitol to remove the protamines in order to form nuclear halos. These were then treated with either restriction endonucleases to release the DNA not directly associated with the nuclear matrix or with DNAse I to digest all the DNA. The treated sperm nuclei were injected into oocytes, and the paternal pronuclear formation and DNA synthesis was monitored. We found that restriction digested sperm nuclear halos were capable of forming paternal pronuclei and initiating DNA synthesis. However, when isolated mouse sperm DNA or sperm DNA reconstituted with the nuclear matrices were injected into oocytes, no paternal pronuclear formation or DNA synthesis was observed. These data suggest that the in situ nuclear matrix attachment organization of sperm DNA is required for mouse paternal pronuclear DNA synthesis.     

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.     

Assembly of correct kinetochore architecture in Xenopus egg extract requires transition of sperm DNA through interphase

Xenopus egg extracts provide a powerful tool for studying the formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one uses a direct chromatin assembly from sperm nuclei in cytostatic factor (CSF)-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step with subsequent reestablishment of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: The amounts of outer kinetochore proteins such as Bub1, BubR1, and Dynactin subunit p150^glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. On the contrary, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results indicate that the transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.     

Daunomycin disrupts nuclear assembly and the coordinate initiation of DNA replication in Xenopus egg extracts

We have used Xenopus egg extracts to investigate the effects of the antitumor drug daunomycin on DNA replication in vitro. Xenopus sperm nuclei replicated nearly synchronously in our egg extracts, thereby allowing us to determine the effects of the drug on both replication initiation and elongation. Titration experiments demonstrated that daunomycin effectively inhibited replication in the extract, with 50% inhibition at a total drug concentration of 2.7 μM. However, a high concentration of daunomycin 150 μM) also inhibited nuclear envelope assembly, a prerequisite for the initiation of replication in this system. Therefore, to bypass the effects of daunomycin on nuclear envelope assembly, sperm nuclei were preassembled in extract prior to drug addition. Initiation of replication in preassembled nuclei was also inhibited by daunomycin, with 50% inhibition at a drug concentration of 3.6 μM. At low drug concentrations, where replication did occur, the synchrony of initiations within individual nuclei was lost. This drug-induced disruption of initiation events may provide important clues regarding the mechanism(s) by which these events are coordinated in eukaryotic cells. Daunomycin also inhibited replication elongation in preassembled, preinitiated nuclei. However, the concentration of drug required for 50% inhibition of elongation was nearly fourfold higher than that required for inhibition of initiation. Taken together, these data demonstrate that Xenopus egg extract can be used to investigate the effects of DNA-binding antitumor drugs on a number of interrelated cellular processes, many of which are less tractable in whole cell systems. J. Cell. Biochem. 64:476–491. © 1997 Wiley-Liss, Inc.     

Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint

A strict control of replication origin density and firing time is essential to chromosomal stability. Replication origins in early frog embryos are located at apparently random sequences, are spaced at close ( approximately 10-kb) intervals, and are activated in clusters that fire at different times throughout a very brief S phase. Using molecular combing of DNA from sperm nuclei replicating in Xenopus egg extracts, we show that the temporal order of origin firing can be modulated by the nucleocytoplasmic ratio and the checkpoint-abrogating agent caffeine in the absence of external challenge. Increasing the concentration of nuclei in the extract increases S phase length. Contrary to a previous interpretation, this does not result from a change in local origin spacing but from a spreading of the time over which distinct origin clusters fire and from a decrease in replication fork velocity. Caffeine addition or ATR inhibition with a specific neutralizing antibody increases origin firing early in S phase, suggesting that a checkpoint controls the time of origin firing during unperturbed S phase. Furthermore, fork progression is impaired when excess forks are assembled after caffeine treatment. We also show that caffeine allows more early origin firing with low levels of aphidicolin treatment but not higher levels. We propose that a caffeine-sensitive, ATR-dependent checkpoint adjusts the frequency of initiation to the supply of replication factors and optimizes fork density for safe and efficient chromosomal replication during normal S phase.     

A simplified method of generating transgenic Xenopus

Currently transgenic frog embryos are generated using restriction-enzyme-mediated integration (REMI) on decondensed sperm nuclei followed by nuclear transplantation into unfertilized eggs. We have developed a simplified version of this protocol that has the potential to increase the numbers of normally developing transgenic embryos.     

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.     

Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs

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.     

Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication

During late mitosis and early G₁, a series of proteins are assembled onto replication origins, resulting in them becoming ‘licensed’ for replication in the subsequent S phase. Four factors have so far been identified that are required for chromatin to become functionally licensed: ORC (the origin recognition complex) and Cdc6, plus the two components of the replication licensing system RLF-M and RLF-B. Here we describe the first steps of a systematic fractionation of Xenopus egg extracts to identify all the components necessary for the assembly of licensed replication origins on Xenopus sperm nuclei (the physiological DNA substrate in this system). We have purified a new activity essential for this reaction, and have shown that it is nucleoplasmin, a previously known chromatin remodelling protein. Nucleoplasmin decondenses the sperm chromatin by removing protamines, and is required at the earliest known step in origin assembly to allow ORC to bind to the DNA. Sperm nuclei can be licensed by a combination of nucleoplasmin, RLF-M and a partially purified fraction that contains ORC, Cdc6 and RLF-B. This suggests that we are likely to have identified most of the proteins required for this assembly reaction.     

Mitotic remodeling of the replicon and chromosome structure

Animal cloning by nuclear-transfer experiments frequently fails due to the inability of transplanted nuclei to support normal embryonic development. We show here that the formation of mitotic chromosomes in the egg context is crucial for adapting differentiated nuclei for early development. Differentiated erythrocyte nuclei replicate inefficiently in Xenopus eggs but do so as rapidly as sperm nuclei if a prior single mitosis is permitted. This mitotic remodeling involves a topoisomerase II-dependent shortening of chromatin loop domains and an increased recruitment of replication initiation factors onto chromatin, leading to a short interorigin spacing characteristic of early developmental stages. It also occurs within each early embryonic cell cycle and dominantly regulates initiation of DNA replication for the subsequent S phase. These results indicate that mitotic conditioning is crucial to reset the chromatin structure of differentiated adult donor cells for embryonic DNA replication and suggest that it is an important step in nuclear cloning.     

Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry

To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 15 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm nuclei were pretreated with dithiothreitol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same six species of sperm nuclei were induced to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear size were monitored by phase-contrast microscopy and flow cytometry. As occurred in the oocyte, human sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be related to the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn, be determined by the type(s) of sperm nuclear protamine(s) present.     

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.     

Induction of DNA synthesis or apoptosis in mammalian nuclei by Xenopus egg extracts that fail to support the replication of sperm chromatin

Cell-free extracts of Xenopus eggs are widely used to study DNA replication in higher eukaryotes. However, with Xenopus sperm chromatin as template, it is a common experience that the efficiency of replication varies from extract to extract, for reasons that are unclear. Here we show that the majority of extracts unable to support sperm replication are nevertheless able to induce DNA synthesis in intact mammalian nuclei. Those that do not, induce apoptosis.     

Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system.

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.     

Fertilization by sperm injection in the rabbit

Whole rabbit spermatozoa and isolated sperm nuclei were microinjected directly into the ooplasm of hamster and rabbit ova. These injected sperm decondensed and formed male pronuclei during subsequent in-vitro culture. Injection of whole spermatozoa and sperm nuclei prepared by a protocol known to allow in-vitro capacitation of ejaculated spermatozoa yielded a significantly higher (P < 0.01) number of activated rabbit ova containing male pronuclei than did injection of uncapacitated epididymal sperm nuclei or ejaculated sperm nuclei. Rabbit ova fertilized by sperm injection were capable of undergoing normal-appearing cleavage division during 22 h of culture.