Site-specific and temporally controlled initiation of DNA replication in a human cell-free system

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G₁ phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G₁ phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G₁ phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.     

Distinct roles for cyclins E and A during DNA replication complex assembly and activation

Initiation of DNA replication is regulated by cyclin-dependent protein kinase 2 (Cdk2) in association with two different regulatory subunits, cyclin A and cyclin E (reviewed in ref. 1). But why two different cyclins are required and why their order of activation is tightly regulated are unknown. Using a cell-free system for initiation of DNA replication that is based on G1 nuclei, G1 cytosol and recombinant proteins, we find that cyclins E and A have specialized roles during the transition from G0 to S phase. Cyclin E stimulates replication complex assembly by cooperating with Cdc6, to make G1 nuclei competent to replicate in vitro. Cyclin A has two separable functions: it activates DNA synthesis by replication complexes that are already assembled, and it inhibits the assembly of new complexes. Thus, cyclin E opens a 'window of opportunity' for replication complex assembly that is closed by cyclin A. The dual functions of cyclin A ensure that the assembly phase (G1) ends before DNA synthesis (S) begins, thereby preventing re-initiation until the next cell cycle.     

Cdc6 protein causes premature entry into S phase in a mammalian cell-free system.

We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.     

Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor

We have investigated the mechanism which prevents reinitiation of DNA replication within a single cell cycle by exploiting the observation that intact G2 HeLa nuclei do not replicate in Xenopus egg extract, unless their nuclear membranes are first permeabilized (Leno et al., 1992). We have asked if nuclear membrane permeabilization allows escape of a negative inhibitor from the replicated nucleus or entry of a positive activator as proposed in the licensing factor hypothesis of Blow and Laskey (1988). We have distinguished these possibilities by repairing permeabilized nuclear membranes after allowing soluble factors to escape. Membrane repair of G2 nuclei reverses the effects of permeabilization arguing that escape of diffusible inhibitors is not sufficient to allow replication, but that entry of diffusible activators is required. Membrane repair has no significant effect on G1 nuclei. Pre-incubation of permeable G2 nuclei in the soluble fraction of egg extract before membrane repair allows semiconservative DNA replication of these nuclei when incubated in complete extract. Addition of the same fraction after membrane repair has no effect. Our results provide direct evidence for a positively acting "licensing" activity which is excluded form the interphase nucleus by the nuclear membrane. Nuclear membrane permeabilization and repair can be used as an assay for licensing activity which could lead to its purification and subsequent analysis of its action within the nucleus.     

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with ³H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.     

Replication of Nuclei from Cycling and Quiescent Mammalian Cells in 6-DMAP-TreatedXenopusEgg Extract

Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei inXenopusegg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [α-³²P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the “licensed state” of G0 nuclei using the assays described here.     

Giardia lamblia: autoradiographic analysis of nuclear replication

Giardia lamblia trophozoites, grown in axenic culture, were labeled for various periods of time with [3H]thymidine. After autoradiography, grains were counted over each of the two nuclei in each trophozoite. Analysis of the fraction of trophozoites labeled for each time period resulted in an estimate of a generation time of 15 hr. The DNA synthetic or S phase for a trophozoite in culture was calculated to be 1.8 hr. G1 and G2 periods were determined to be 8.5 and 3 hr, respectively. A comparison of the labeling density between the two nuclei indicated that replication takes place simultaneously in both nuclei for at least 70% of S period. The fraction of asymmetrically labeled trophozoites is consistent with a model in which the nuclei replicate out of phase by 15-30 min, but, due to the small diameter of the nuclei relative to the grain size, the possibility that replication takes place simultaneously in both nuclei of a trophozoite throughout the S phase cannot be ruled out.     

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

Summary Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluroescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluores-cence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase is consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.     

DNA replication in quiescent cell nuclei: regulation by the nuclear envelope and chromatin structure

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H1(0) are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.     

A limited number of chromosomes makes a nucleus competent to respond to inducers of replication and mitosis in a plant.

Multinucleate tetraploid cells with unbalanced chromosomal distribution in aneuploid nuclei were obtained in Allium cepa L. root meristems. For this, their natural diploid cells were treated with a multipolarizing agent (1 h carbetamide) followed by an inhibitor of cytokinesis (1 h caffeine). Data from these multinucleate cells with aneuploid nuclei suggest that only four out of the thirty-two chromosomes of their autotetraploid complement possess DNA sequences making the nucleus competent to respond to inducers of replication and mitosis. Direct observation of cells where a single replicated chromosome had reached mitosis showed that this chromosome was the one bearing the nucleolar organizer. Six specific chromosomes would confer competence to the nucleus to respond to inducers of replication but not to those producing chromosome condensation. Another four different chromosomes would confer the nucleus with the ability to respond to mitotic inducers but not to replication inducers. The rest of the chromosomal complement seemed to lack any of the DNA sequences needed for these two important cycle transitions. In a nutshell, certain DNA sequences distributed in a few chromosomes of the onion complement are an intranuclear requirement to initiate replication and mitosis in these plant cells.     

The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts

We have exploited a property of chicken erythrocyte nuclei to analyze the regulation of DNA replication in a cell-free system from Xenopus eggs. Many individual demembranated nuclei added to the extract often became enclosed within a common nuclear membrane. Nuclei within such a "multinuclear aggregate" lacked individual membranes but shared the perimeter membrane of the aggregate. Individual nuclei that were excluded from the aggregates initiated DNA synthesis at different times over a 10-12-h period, as judged by incorporation of biotinylated dUTP into discrete replication foci at early times, followed by uniformly intense incorporation at later times. Replication forks were clustered in spots, rings, and horseshoe-shaped structures similar to those described in cultured cells. In contrast to the asynchronous replication seen between individual nuclei, replication within multinuclear aggregates was synchronous. There was a uniform distribution and similar fluorescent intensity of the replication foci throughout all the nuclei enclosed within the same membrane. However, different multinuclear aggregates replicated out of synchrony with each other indicating that each membrane-bound aggregate acts as an individual unit of replication. These data indicate that the nuclear membrane defines the unit of DNA replication and determines the timing of DNA synthesis in egg extract resulting in highly coordinated triggering of DNA replication on the DNA it encloses.     

Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts

To investigate how the protein kinase cdc7 stimulates DNA replication in metazoans, a soluble cell-free replication system derived from Xenopus eggs was used. DNA was incubated in egg cytosol to form prereplication complexes and then in nucleoplasmic extract to initiate DNA synthesis. We find that cdc7 is greatly enriched in nucleoplasmic extract and that this high concentration is essential for efficient DNA replication, supporting previous models that the nucleus activates replication indirectly by sequestering essential components. cdc7 binds to chromatin at the G(1)/S transition before initiation occurs, and it dissociates from chromatin as S phase progresses. The chromatin association of cdc7 requires chromatin-bound MCM. In turn, cdc7 is required to load the initiation factor cdc45 onto the DNA. Finally, efficient replication is observed when chromatin is exposed first to cdc7 and then to cdk2 but not when it is exposed to cdk2 before cdc7. Therefore, the cdc7- and cdk2-dependent initiation steps can be separated, indicating the existence of a novel, stable initiation intermediate. Moreover, the data suggest that cdk2 can only act after cdc7 has executed its function.     

Asynchronous DNA replication and origin licensing in the mouse one‐cell embryo

To prevent duplicate DNA synthesis, metazoan replication origins are licensed during G1. Only licensed origins can initiate replication, and the cytoplasm interacts with the nucleus to inhibit new licensing during S phase. DNA replication in the mammalian one‐cell embryo is unique because it occurs in two separate pronuclei within the same cytoplasm. Here, we first tested how long after activation the oocyte can continue to support licensing. Because sperm chromatin is licensed de novo after fertilization, the timing of sperm injection can be used to assay licensing initiation. To experimentally skip some of the steps of sperm decondensation, we injected mouse sperm halos into parthenogenetically activated oocytes. We found that de novo licensing was possible for up to 3 h after oocyte activation, and as early as 4 h before DNA replication began. We also found that the oocyte cytoplasm could support asynchronous initiation of DNA synthesis in the two pronuclei with a difference of at least 2 h. We next tested how tightly the oocyte cytoplasm regulates DNA synthesis by transferring paternal pronuclei from zygotes generated by intracytoplasmic sperm injection (ICSI) into parthenogenetically activated oocytes. The pronuclei from G1 phase zygotes transferred into S phase ooplasm were not induced to prematurely replicate and paternal pronuclei from S phase zygotes transferred into G phase ooplasm continued replication. These data suggest that the one‐cell embryo can be an important model for understanding the regulation of DNA synthesis. J. Cell. Biochem. 107: 214–223, 2009. © 2009 Wiley‐Liss, Inc.     

A Protein Kinase-Dependent Block to Reinitiation of DNA Replication in G2 Phase in Mammalian Cells

Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation inXenopusegg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations byXenopusegg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitorsin vivo.Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied byXenopusegg extract, which are distinct from those required for replication licensing.     

Uncoupling global and fine-tuning replication timing determinants for mouse pericentric heterochromatin

Mouse chromocenters are clusters of late-replicating pericentric heterochromatin containing HP1 bound to trimethylated lysine 9 of histone H3 (Me3K9H3). Using a cell-free system to initiate replication within G1-phase nuclei, we demonstrate that chromocenters acquire the property of late replication coincident with their reorganization after mitosis and the establishment of a global replication timing program. HP1 dissociated during mitosis but rebound before the establishment of late replication, and removing HP1 from chromocenters by competition with Me3K9H3 peptides did not result in early replication, demonstrating that this interaction is neither necessary nor sufficient for late replication. However, in cells lacking the Suv39h1,2 methyltransferases responsible for K9H3 trimethylation and HP1 binding at chromocenters, replication of chromocenter DNA was advanced by 10-15% of the length of S phase. Reintroduction of Suv39h1 activity restored the later replication time. We conclude that Suv39 activity is required for the fine-tuning of pericentric heterochromatin replication relative to other late-replicating domains, whereas separate factors establish a global replication timing program during early G1 phase.     

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.     

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.     

Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner

The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.     

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.     

Chromosome replication in cell-free systems from Xenopus eggs

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 semi-conservation 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.