Cell, Vol. 69,

151-158,

April 3, 1992, Copyright 0 1992 by Cell Press

The Nuclear Membrane Prevents Replication of Human G2 Nuclei but Not Gl Nuclei in Xenopus Egg Extract Gregory H. Leno,*t C. Stephen Downes,tS and Ronald A. Laskey’t *Wellcome/CRC Institute of Cancer and Developmental Biology University of Cambridge Tennis Court Road Cambridge CB2 1QR England *CRC Mammalian DNA Repair Group tDepartment of Zoology University of Cambridge Downing Street Cambridge CB2 3EJ England

Summary We have used synchronized HeLa cells to investigate the role of the nuclear membrane in preventing rereplication in a single cell cycle. Nuclei were prepared with intact nuclear membranes using streptolysin-0 or dig itonin and assayed for replication in Xenopus egg extracts. Intact Gl nuclei replicate semiconservatively, but intact G2 nuclei do not replicate in egg extract. However, permeabilizing the nuclear membranes of G2 nuclei by treatment with NP-40 allows them all to replicate in egg extract under cell cycle control, suggesting that integrity of the nuclear membrane is required to distinguish G2 from Gl human nuclei and to prevent rereplication within a single cell cycle. The results are discussed in terms of the previously proposed licensing factor model. Introduction DNA replication in eukaryotic cells is a precisely controlled process. Within a single S phase, all genomic DNA must be replicated once and only once prior to the onset of mitosis. If any DNA fails to replicate or if any rereplication of DNA occurs within a single S phase, abnormal mitosis or DNA amplification will result. As these events are rare, it is clear that the processes that coordinate initiation of replication at thousands of sites within the genome are precisely regulated to prevent underreplication or overreplication. Evidence of a regulatory mechanism that prevents repeated replication of replicated DNA came from the cell fusion experiments of Rao and Johnson (1970). These studies showed that Gl and G2 nuclei differ in their capacities for DNA replication. Gl nuclei were induced into premature S phase following fusion of Gl with S phase cells, whereas G2 nuclei did not initiate DNA replication following fusion of G2 and S phase cells. In addition, neither Gl nor G2 cytoplasmic components inhibited DNA synthesis in nuclei of S phase cells when these cells were fused with Gl or G2 cells, suggesting that Gl and G2 nuclei differ in

some fundamental way regarding their replication capacity. Insight into the nature of the regulatory processes involved has come from studies using a cell-free system, derived from the eggs of Xenopus laevis. This cell-free system initiates and completes DNA replication efficiently, under cell cycle control. Using amphibian egg extracts, it has become increasingly clear that initiation of DNA replication is dependent on nuclear structure and that regulation of DNA replication is coupled to assembly of an intact nucleus (Lohka and Masui, 1983,1984; Blow and Laskey, 1986; Newport, 1987; Blow and Watson, 1987; Sheehan et al., 1988; Blow and Sleeman, 1990; Leno and Laskey, 1991). One specific feature of nuclear structure, namely the nuclear membrane, has been implicated in the mechanism that prevents repeated DNA replication within a single cell cycle (Blow and Laskey, 1988). Nuclear proteins contain nuclear localization sequences, which allow them to enter the nucleus through the nuclear pore complex, while cytoplasmic proteins are excluded. This laboratory has previously shown that disrupting the selective permeability of the nuclear envelope of a replicated Xenopus sperm nucleus by permeabilizing it with lysolecithin is sufficient for its rereplication in Xenopus egg extract, without passage through mitosis. This observation can be explained by a model in which an essential replication factor is unable to enter the nucleus because it lacks a functional nuclear localization sequence. Therefore it would bind to DNA only at mitosis when the nuclear membrane is broken down, and it would be destroyed after replication initiates. In this way, the factor would license the DNA to replicate once and only once following nuclear reassembly, but further replication could not occur until the nuclear membrane breaks down again in the next mitosis (Blow and Laskey, 1988). However, the cell cycles of early Xenopus embryos are highly abnormal, lasting only 35 min and consisting of alternating S and M phases, without Gl or G2 phases. Therefore, a key question is whether or not nuclear membrane integrity is required to distinguish the different replication capabilities of Gl and G2 mammalian nuclei. In this report, we have used the Xenopus cell-free replication system to investigate the role of the nuclear membrane in defining the replication capacity of human somatic cell nuclei isolated from different stages within the cell cycle. We have used the detergent digitonin, or the bacterial exotoxin streptolysin-0 (SLO), to isolate nuclei with intact nuclear membranes from HeLa cells synchronized in Gl and G2 of the cell cycle. We have also permeabilized these Gl and G2 nuclei with Nonidet P-40 (NP-40) in order to assess any difference in replication capacity, between intact and permeabilized nuclei, in Xenopus egg extract. Nuclear membrane permeability was determined by incubating either detergent- or SLO-treated HeLa cells with TRITC-immunoglobulin G (IgG). We report here that intact Gl nuclei replicate semiconservatively in egg ex-

tract. In striking contrast, intact G2 nuclei do not replicate in this system. However, when the nuclear membrane of an intact G2 nucleus is permeabilized by NP-40, then G2 nuclei also replicate undercell cycle control. Thissuggests that nuclear membrane integrity may be important for limiting DNA replication to a single round per cell cycle in human cells. Results Isolation of Intact Gl and 62 HeLa Nuclei The major aim of this study was to determine what role, if any, the nuclear membrane from somatic human cells plays in distinguishing a replicated (G2) nucleus from an unreplicated (Gl) nucleus. To address this question, it was essential to prepare nuclei, with intact nuclear membranes, from cells synchronized in Gl and G2 of the cell cycle. Nuclei prepared by conventional methods using mechanical shear, NP-40, or lysolecithin have damaged nuclear membranes that are no longer selectively permeable to only nuclear proteins (see below). We used two procedures to prepare nuclei with intact membranes. Initially, HeLa cells were synchronized in Gl and G2 of the cell cycle as described in the Experimental Procedures. In the first procedure, cells from each population were then treated with the detergent digitonin. Digitonin binds membrane-associated cholesterol and has been used to permeabilize the plasma membrane of cultured cells for analysisof avariety of cellular processes (Wilson and Kirshner, 1983; Peppers and Holz, 1986; Sarafian et al., 1987; Bittner and Holz, 1988; Lazarovici et al., 1989). In addition, Adam et al. (1990) have recently used digitonin to isolate intact nuclei from HeLa cells. In the second procedure, we used SLO, a 69 kd streptococcal toxin that also binds membrane-associated cholesterol (Duncan and Schlegel, 1975; Prigent and Alouf, 1976; Alouf, 1980), to permeabilize the plasma membrane selectively. However, its mechanism of membrane permeabilization differs greatly from that of the detergent digitonin. In this case, following binding of SLO monomer to cholesterol, these protein-cholesterol complexes associate via lateral diffusion along the membrane (Hugo et al., 1986) and form oligomers which, when inserted into the membrane, form barrel-stave pores up to 35 nm in diameter (Ojcius and Young, 1991). One key feature of this approach is that SLO binding can be dissociated from pore formation by lowering the temperature to 0% (Hugo et al., 1986). At this temperature, SLO monomer binds to the membrane; but pore formation does not occur until the temperature is increased (Hugo et al., 1986; Ahnert-Hilger et al., 1989b), thus allowing the selective permeabilization of the plasma membrane. These pores, apparently similar in size to those produced by digitonin treatment (AhnertHilger et al., 1989a), are sufficient to allow movement of antibodies into the cell (Ahnert-Hilger et al., 1989b), whereas antibodies (e.g., IgG) do not enter the nucleus through nuclear pores (Paine, 1975) unless nuclear membrane integrity is breached. Therefore, we have used TRITC-IgG as a tool to assay both plasma and nuclear

membrane integrity following treatment of HeLa cells with digitonin or SLO. Figure 1 shows the results of an incubation of TRITCIgG with Gl (A) or G2 (6) HeLa cells that had been treated with SLO to permeabilize their plasma membranes but not their nuclear membranes. A bright cytoplasmic fluorescence was observed in virtually all cells, indicating that the plasma membranes were permeable to IgG. However, a general fluorescence was not observed in the nuclei of these cells, demonstrating that their nuclear membranes were impermeable to IgG and indicating that nuclear membrane integrity within these cells had not been altered by SLO treatment. Identical results were obtained following incubation of digitonin-permeabilized Gl and G2 HeLa cells with TRITC-IgG (data not shown), demonstrating that either protocol can be used to prepare permeabilized HeLa cells with intact nuclei. The permeabilized cells prepared by these protocols will be referred to as “nuclei” since they are virtually intact nuclei with some cytoplasmic material still attached. The intense nucleolar fluorescence observed in both Gl and G2 nuclei (Figure 1) probably reflects the association of contaminating free fluorochrome with these organelles, because it varies between different batches of TRITCIgG, even when these batches are added to the same nuclear preparation, and becausefreefluorochrome alone is sufficient to produce this pattern of nucleolar fluores-

SLO

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Figure 1. Preparation chronized HeLa Cells

of Intact

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HeLa cells were synchronized in Gi and G2 of the cell cycle. Intact Gl and G2 nuclei were prepared by disruption of the plasma membrane with SLO (A, 6). The nuclear membranes were subsequently permeabilized in aliquots of these cells by treatment with NP-40 (C, D). The integrity of the nuclear membrane was assayed by incubation with TRITC-IgG, and rhodamine fluorescence was examined by confocal microscopy. Treatment of cells with SLO resulted in a strong cytoplasmic fluorescence but no uniform nuclear fluorescence, indicating plasma membrane disruption without permeabilization of the nuclear membrane (Gl-SLOIGBSLO). Treatment of unpermeabilized nuclei with NP-40 resulted in uniform cytoplasmic and nuclear fluorescence, indicating both plasma and nuclear membrane permeabilization (GlNP40/G2-NP40). (Evidence that nucleolar fluorescence in [A] and [B] is due to free rhodamine is presented in the text.)

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cence. Therefore, from Figure 1 and similar experiments we conclude that both Gl and G2 nuclei, prepared by SLO or digitonin, have intact nuclear membranes that exclude nonnuclear proteins. To address whether nuclear membrane integrity is important in regulating replication, it was necessary to permeabilize the nuclear membranes of Gl and G2 nuclei and assess their replication capacity in egg extract. Permeabilized nuclei were prepared from intact nuclei, which had been prepared by SLO or digitonin, by subsequent treatment with NP-40 (see Experimental Procedures). Figure 1 shows fields of NP40-treated Gl (C) and G2 (D) nuclei incubated with TRITC-IgG. In striking contrast to the appearance of nuclei in SLO-treated cells (Figures 1A and lB), a clear uniform nuclear fluorescence was observed in >99% of nuclei from both cell populations (Figures 1C and lD), demonstrating that the nuclear membranes were permeable to IgG following this treatment. In addition to this uniform fluorescence, the nucleolar fluorescence seen in SLO-prepared nuclei was still observed. The intense cytoplasmic fluorescence seen in both the SLO-treated cells (Figures 1A and 1 B) and digitonintreated cells (data not shown) may reflect nonspecific binding of the TRITC-IgG to the cytoplasmic components that remain following these mild preparation procedures but that are nearly absent from the NP40-treated cells (Figures 1 C and 1 D). Intact Gl Nuclei Replicate in Egg Extract; Intact G2 Nuclei Do Not To determine whether intact Gl or G2 HeLa nuclei, prepared with SLO or digitonin, were capable of replicating in Xenopus egg extract, we incubated nuclei at 3 ng of DNA per ~1 of extract for 6 hr in the presence of biotin-dUTP. Nuclei were subsequently fixed with ethylene glycol bis (succinimidyl succinate) (EGS), spun onto polylysinecoated coverslips, and stained with Hoechst 33258 for total DNA (Hoechst) and fluorescein-conjugated streptavidin (Biotin). The streptavidin binds to the biotinylated precursor, which is incorporated into nascent DNA. The extent of biotin incorporation and hence streptavidin fluorescence is proportional to nascent DNA content and thus illustrates the extent of replication of each nucleus (Blow and Watson, 1987; Leno and Laskey, 1991). Figure 2 shows fields of SLO-prepared Gl and G2 HeLa nuclei stained with Hoechst (A and D) and fluorescein streptavidin (B and E) following incubation in egg extract. All Gl nuclei in Figure 28 show streptavidin fluorescence, indicating incorporated biotin-dUTP; however, in striking contrast, only 2 of 11 nuclei from the G2 population of cells shown in Figure 2E have incorporated biotin-dUTP. The fields shown in Figure 2 are representative of each population of nuclei incubated in egg extract. This is illustrated in the histogram in Figure 2C. One hundred nuclei, selected at random from each population, were classified as either biotin positive (replicating) or negative (not replicating) based upon the presence or absence, respectively, of streptavidin fluorescence. Ninety-eight percent of Gl nuclei replicated in egg extract; however, only 30% of nuclei

Gl -SLO

Figure 2. Replication Egg Extract

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Unpermeabilized Gl and G2 HeLa nuclei, prepared by SLO treatment, were incubated in egg extract for 6 hr with biotin-dUTP. The G2 cells were pulse-labeled with SrdU during the last 15 min of the cell synchronization protocol to label S phase contaminants. Followmg incubation of Gi and G2 nuclei in extract, nuclei were fixed and spun onto polylysine-coated coverslips as described in the Experimental Procedures Nuclei were stained with Hoechst 33258 for total DNA (Hoechst; [A], [D]) and fluorescein streptavidin for incorporated biotin (Siotin; [S], [El). In addition, G2 nuclei were incubated with anti-SrdU monoclonal antibody and stained with sheep anti-mouse Texas red whole antibody for ErdU incorporated in vivo (ErdU; IF]). One hundred nuclei selected at random from each incubation were classified as showing fluorescein fluorescence (biotin) and/or Texas red (SrdU) fluorescence or as negative (see histogram, [Cl). Ninety-eight percent of the biotin-labeled G2 population were also labeled with SrdU. demonstrating that they were in late S phase and that 2% of true G2 nuclei replicated in egg extract.

from the G2 population replicated in a parallel incubation. This difference is greatly magnified by the fact that, of the 30% of G2 nuclei replicating in the extract, 98% were S phase contaminants of the G2 population. This was determined by pulse-labeling G2 HeLa cells with bromodeoxyuridine (BrdU) for 15 min prior to nuclear preparation with SLO or digitonin. Only those cells in S phase at the time of labeling would incorporate BrdU, and these nuclei were subsequently identified by indirect immunofluorescence using an anti-BrdU monoclonal antibody (Becton Dickinson) and a Texas red-conjugated sheep anti-mouse whole antibody (Amersham). Thirty-five percent of the nuclei from theG2 population of cells were S phase contaminants as illustrated in Figure 2C. To determine whether the nuclei from the G2 population that replicated in egg extract were actually S phase contaminants, we incubated BrdUpulsed G2 nuclei in egg extract with biotin-dUTP for 6 hr. Fixed nuclei were subsequently stained for incorporated BrdU as described above and with fluorescein streptavidin. A typical result is shown in Figure 2F. In this case, 3

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of 11 nuclei have incorporated BrdU, indicating that they are S phase contaminants that were undergoing replication during the 15 min labeling period prior to nuclear isolation. Only 2 of these 3 nuclei also incorporated biotin following incubation in egg extract (Figure2E) indicating that some S phase contaminants in the G2 population exit from S during the in vivo pulse with BrdU. Of 100 biotin-positive nuclei counted, 98% also showed BrdU fluorescence, demonstrating that these nuclei were in S phase at the time of isolation and moreover demonstrating that 2% of genuine G2 nuclei replicated in the egg extract in contrast to the 98% of Gl nuclei. Seven percent of cells, from a population synchronized in Gl, were found to be S phase contaminants, as judged by incorporation of BrdU during a 15 min pulse just prior to nuclear preparation. Results similar to those described above were also observed using nuclei obtained from digitonin-treated cells (data not shown). However, one difference between digitonin- and SLO-treated cells was observed during incubation in egg extract. With some batches of digitonin-treated cells, the plasma membranes were resealed very quickly following addition to the egg extract (data not shown). This was not observed with SLO-treated cells, which may be due to the different mechanism of membrane permeabilization with SLO. We next conducted density substitution experiments to determine whether the incorporated biotin observed in intact Gl nuclei (Figure 28) was due to semiconservative DNA replication; and if so, whether replication was occurring under cell cycle control. Intact Gl nuclei, at 3 ng of DNA per ~1, were incubated for 6 hr in egg extract supplemented with 100 pCi/ml [a-3’P]dATP and 0.25 mM bromodeoxyuridine triphosphate (BrdUTP). As a control, extract was incubated without added HeLa nuclei to detect replication of any endogenous template. The DNA from each sample was isolated and centrifuged to equilibrium in a cesium chloride gradient. The results are shown in Figure 3. With intact Gl nuclei (open circles), asingle peak of [3ZP]dATP incorporation was observed at a density of 1.762 g/cm3, indicating a single complete round of semiconservative DNA replication in egg extract (i.e., heavy/ light DNA; HL). No significant incomplete strand synthesis or rereplication of DNA was Observed. The expected density of rereplicated DNA (ml .79 g/cm3; heavy/heavy DNA; HH) and the density of unreplicated DNA (~1.72 g/cm3; light/light DNA; LL) are indicated in Figure 3. Only background incorporation was observed in the “No DNA” control, i.e., without added HeLa nuclei (closed squares). These results demonstrate that intact Gl HeLa nuclei undergo a single complete round of semiconservative DNA replication in egg extract. Permeabilizing the Nuclear Membrane with NP-40 Allows Replication of G2 Nuclei in Egg Extract To determine whether nuclear membrane integrity was involved in the block to replication observed in intact G2 nuclei, we permeabilized intact Gl and G2 nuclei with NP-40 and incubated both intact and permeabilized nuclei in the egg extract for 6 hr with biotin-dUTP. During incubation in extract, nuclear membranes are reformed around

1

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intact, SLO-prepared Gi nuclei at *3 ng of DNA per fd were incubated in egg extract containing 100 nCi/ml [aJ2P]dATP and 0.25 mM ErdUTP for 6 hr. In a parallel incubation, no HeLa DNA was added to extract as a control for replication of endogenous template. The purified DNA was separated by centrifugation to equilibrium in a cesium chloride density gradient, and collected fractions were spotted onto Whatman GF-C filters. Free label was removed by washing filters and CPM determined by scintillation counting. GI-SLO, open circles; no DNA, closed squares; light/light DNA, LL; heavy/light DNA, HL; heavy/heavy DNA, HH.

most of the NP40-permeabilized nuclei within 2 hr as judged by TRITC-IgG exclusion from the nucleus (data not shown). Nuclei were fixed, spun onto coverslips, and stained with Hoechst 33258 and fluorescein streptavidin. Figure 4 shows a field of intact G2 nuclei (GBSLO) and a field of NP40-permeabilized G2 nuclei (G2-NP40) stained with Hoechst (A and C) and fluorescein streptavidin (6 and D) after incubation in egg extract. Ninety-eight percent of all permeabilized G2 nuclei replicated in egg extract (D) while only 30% of intact G2 nuclei replicated under the same conditions (B); this demonstrates that permeabilizing the nuclear membranes of G2 nuclei with NP-40 allows replication in Xenopus egg extract. In addition, 97% of all NP40-permeabilized Gl nuclei also replicated in the egg extract, demonstrating that replication-competent Gl nuclei are not rendered incompetent for replication by the NP-40 treatment. Intact nuclei, prepared by digitonin treatment, were also permeabilized with NP-40 and incubated in egg extract. The extent of replication observed in these nuclei (data not shown) was similar to that described for SLO-prepared nuclei (Figure 4). We have shown in Figures 2 and 4 that there are clear differences in the replication capacity of unpermeabilized and permeabilized Gl and G2 HeLa nuclei following incubation in egg extract. However, to assess the extent of replication quantitatively, we incubated these nuclei at 3 ng of DNA per t.d of extract for 6 hr with [a-32P]dATP. Samples were stopped, and the DNA was precipitated with trichloroacetic acid. Figure 5 shows the results from a typical set of incubations; the extent of replication is expressed as nanograms of DNA synthesized per microliter of extract. There is a striking difference in the extent of replication between Gl and G2 SLO-prepared nuclei. Eighty per-

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with only residual synthesis in late S phase nuclei that are contaminants of the G2 population (see Figures 2E and 2F). In addition, G2 nuclei with permeabilized nuclear membranes were replicated extensively in egg extract (83%), confirming the biotin data in Figure 4. The extent of replication of permeabilized G2 nuclei in egg extract was similar to permeabilized or unpermeabilized Gl nuclei (90% and 80%, respectively). These data illustrate quantitatively the two key differences between unpermeabilized and permeabilized Gl and G2 HeLa nuclei replicating in egg extract. First, unpermeabilized Gl nuclei replicate in egg extract, but unpermeabilized G2 nuclei do not. Second, permeabilizing the nuclear membranes of G2 nuclei removes the block to rereplication in this system.

G2-NP40

in S)

Figure 4. Permeabilizing the Nuclear Replication in Egg Extract

98% Membranesof

% Nuclei Biotin (+) G2 Nuclei Allows

Intact Gl and G2 nuclei and nuclei from the same populations subsequently permeabilized with NP-40 were incubated in egg extract for 6 hr, fixed, and processed as described in the Experimental Procedures. Nuclei were stained with Hoechst 33256 for total DNA (Hoechst; [A], [Cl) and fluorescein streptavidin for nascent DNA (Biotin; [B], [D]). Typical fields of G2 nuclei, which were either unpermeabilized (G2SLO; [A], [B]) or permeabilzed (G2-NP40; [Cl, [D]) before incubation in egg extract, are shown here. Approximately 96% of the NP-40permeabilized G2 nuclei replicated in egg extract; however, in contrast, ~5% of G2 nuclei replicated in egg extract without prior membrane permeabilization.

cent of the DNA from Gl nuclei was replicated within 6 hr, while only 3% of the DNA from nuclei derived from the G2 population of cells was replicated. This low level of incorporation in the G2 population appears, at first, to be in conflict with the finding that 30% of the population incorporated biotin (see Figure 2C); however, it is consistent

Permeabilized G2 Nuclei Replicate Semiconservatively, under Cell Cycle Control, in Egg Extract Do permeabilized G2 nuclei replicate semiconservatively in egg extract, and if so, is replication restricted to a single round? To address these questions, we conducted density substitution experiments similar to those described in Figure 3. In these experiments, intact G2 nuclei and nuclei subsequently permeabilized with NP-40 were incubated at 3 ng of DNA per WIfor 6 hr in egg extract supplemented with the dense precursor BrdUTP and [a-3ZP]dATP. Substituted DNA was separated by centrifugation to equilibrium in a cesium chloride gradient. Figure 6 shows the peaks

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Figure 5. Quantitation ized Gi and G2 HeLa

NP40

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Intact (SLO) and permeabilized (NP40) Gl and G2 HeLa nuclei at -3 ng of DNA per trl of extract were incubated in egg extract containing 100 rrCi/ml [aJZP]dATP for 6 hr. Reactions were stopped, and the DNA was precipitated with trichloroacetic acid. DNA replication is expressed as nanograms of DNA synthesized per microliter of extract.

G2 HeLa Nuclei Replicate Control rn Egg Extract

Semiconserva-

Both intact (SLO) and permeabilized (NP-40) G2 nuclei were incubated at 3 ng of DNA per ~1 of extract in egg extract supplemented with 0.25 mM BrdUTP and 100 hCi/ml [a-32P]dATP for 6 hr. The DNA was purified and centrifuged to equilibrium in a cesium chloride density gradient. Fractions were collected and spotted onto Whatman GF-C filters. DNA was precipitated with trichloroacetic acid, and free label was removed by washing filters. Counts per minute (CPM) were determined by scintillation counting. (A) GP-SLO; (B) G2-NP40. Light/light DNA, LL; heavy/light DNA, HL; heavy/heavy DNA, HH

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of 32P incorporation for unpermeabilized G2 nuclei ([A]; GBSLO) and for G2 nuclei permeabilized with NP-40 ([B]; G2-NP40). Very little incorporation of 3ZP is observed with unpermeabilized G2 nuclei (A), consistent with the data shown in Figure 5. A small peak at a density of 1.762 g/cm3 can be seen with an additional plateau of incorporation continuing to the position of unsubstituted DNA (light/light DNA; LL). This plateau of incorporation indicates incomplete strand synthesis and is consistent with residual synthesis in late S phase nuclei present in the G2 population. In contrast, a single prominent peak of 32P incorporation is observed at a density of 1.762 g/cm3 in permeabilized G2 nuclei (B), indicating a single, complete round of DNA replication (heavy/light DNA; HL). No significant rereplication of DNA was observed. Rereplicated DNA would appear as a peak of incorporation at a density of ~1.79 g/cm3 (heavy/heavy DNA; HH). Taken together, these data illustrate two important points. First, following nuclear membrane permeabilization, G2 nuclei replicate semiconservatively in the egg extract, and second, these nuclei replicate under strict cell cycle control, being limited to one round of replication in extract. We discuss below the significance of the observation that only one round of replication follows permeabilization of nuclei. Discussion We have investigated whether or not nuclear membrane integrity contributes to the different replication capacities of Gl and G2 HeLa nuclei reported by Rao and Johnson (1970). We have shown that Gl nuclei, with intact, unpermeabilized nuclear membranes, undergo efficient semiconservative DNA replication once per cell cycle in a Xenopus egg extract. In contrast, intact, unpermeabilized G2 nuclei are not replicated in egg extract under identical conditions. Only S phase contaminants of the G2 cell population continue replication. However, if the nuclear membranes of intact G2 nuclei are permeabilized before addition to extract, the nuclei subsequently undergo a single round of semiconservative replication. Our results are consistent with earlier cell fusion studies showing that Gl nuclei enter S phase prematurely following fusion of Gl HeLa cells with either S phase cells (Rao and Johnson, 1970) or erythrocyte ghosts loaded with S phase extracts (Brown et al., 1985); and that G2 HeLa nuclei are unable to reenter S phase following fusion of G2 and S phase cells (Rao and Johnson, 1970), in spite of the presence of all the factors required for DNA synthesis. These results demonstrate that some fundamental difference exists between the replication capacity of pre- and post-S phase nuclei. Our observation that the nuclear membrane serves as a block to rereplication of G2 HeLa nuclei in egg extract suggests that the nuclear membrane may also limit DNA replication to a single round per cell cycle in human cells and may explain the observations of Rao and Johnson (1970). De Roeper et al. (1977) microinjected Gl and G2 HeLa nuclei, obtained from ruptured cells, into Xenopus eggs and found that although both Gl and G2 nuclei swelled,

a greater number of Gl nuclei than G2 nuclei incorporated ‘H-thymidine. However, they were unable to observe this difference when the nuclei were prepared by a standard detergent lysis procedure. The experiments of De Roeper et al. (1977) are entirely consistent with our observations, and we believe that they can now be interpreted as supporting a role for the nuclear membrane in preventing rereplication. Permeabilizing the nuclear membrane by detergent allows one complete round of replication by G2 nuclei, but it does not allow more than one (Figure 6). At first sight this may seem surprising. Why don’t the holes in the membrane allow continuous access of a licensing factor and thus continued reinitiation? In fact, the observation of only one round of replication is entirely consistent with other information on replication in egg extracts, namely that after nuclear membrane permeabilization, a nuclear membrane reforms, using lipids from the extract, and that initiation only occurs after the membrane has reformed (Newport, 1987; Sheehanet al., 1988; Blowand Sleeman, 1990; Leno and Laskey, 1991). Since initiation does not occur until the nuclear membrane has reformed in the extract, permeabilization should indeed lead to only one round of replication as seen in Figure 6. There are several possible mechanisms by which an intact nuclear membrane may prevent rereplication of human G2 nuclei in egg extract. In theory, the nuclear membranecould function to exclude a positive“licensing”factor from the nucleus as suggested by Blow and Laskey (1988) or to prevent diffusion of a negative inhibitor out of the nucleus until nuclear membrane breakdown at mitosis. However, a diffusible negative factor seems unlikely, because it might then bind to unreplicated DNA, preventing completion of replication. For this reason we continue to prefer the positive “licensing factor” as the simplest alternative, but we are aware that more complicated variants involving both positive and negative components could also explain the data. Support for the positive licensing factor model has recently come from the unexpected direction of the yeast Saccharomyces cerevisiae. Although the yeast nuclear membrane does not break down in mitosis, a protein that is required for initiation of replication remains in the cytoplasm until mitosis, when it enters the nucleus (Hennessy et al., 1990). It is encoded by the CDC46 gene, and it shows sequence homology to several other gene products including yeast Mcm2 and Mcm3 (Yan et al., 1991) and another protein from mouse cells (Hennessy et al., 1991). The behavior of CDC46 precisely resembles that predicted for the hypothetical licensing factor. Therefore, it will be interesting to determine if any of this family of related proteins functions as a licensing factor to allow one and only one round of replication in each cell cycle. We have shown here that integrity of the nuclear membrane is required to distinguish between the replication capabilities of Gl and G2 human cells. It will be interesting to determine whether this is due to exclusion of a protein resembling the CDC46 gene product functioning as a positive licensing factor in human cells.

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of G2 Nuclei

Procedures

Preparation of Egg Extracts Xenopus egg extracts were prepared essentially according to Leno and Laskey (1991) with the following modifications. The protease inhibitors leupeptin, pepstatin A, and aprotinin, all at 10 uglml, were included in the final rinse with extraction buffer. In addition, the eggs were packed by centrifugation at 1500 rpm for 2 min, and all excess buffer was removed prior to the crushing spin. Extracts were supplemented with glycerol to 2% and frozen as beads in liquid nitrogen. Cell Culture and Synchronization HeLacellsweregrown in monolayerculturein 150mmplasticdishesat 37OC in Earle’s minimal essential medium (Gibco, Bethesda Research Laboratories) supplemented with 25% fetal calf serum and 2.5% newborn calf serum (ICN Flow) and with 60 U/ml penicillin and 50 uglml streptomycin. Populations of cells in Gl were obtained by release of mitotically arrested HeLa. These were synchronized by an initial S phase block for 20 hr with 2.5 mM thymidine, followed after a 5 hr interval by a 9 hr mitotic block with high pressure nitrous oxide (5 atm), using the automatic synchronizing apparatus of Downes et al. (1987). Mitotic cells were selectively shaken off, and the mitotic index was checked in cytocentrifuge preparations; the index was 95% or higher. Mitotic populahons were plated out and allowed 3 hr to proceed into Gl, when nuclear preparations were made; we have routinely found that our strain of HeLa is still in early Gl at this time, being postanaphase but not yet incorporating labeled thymidine (Downes et al., 1979; Jost and Johnson, 1981; Downes and Collins, 1982). To detect any S phase contaminants in the present experiments, cultures were given a 15 min pulse of 100 uM BrdU immediately before nuclear preparations were made. Populations of cells enriched for G2 were obtained by release from a double thymidine block (Rao and Johnson, 1970). After 17 hr of initial S phase block with 2.5 mM thymidine, cells were released into fresh medium for 9 hr, then given a second thymidine block for 15 hr; this protocol accumulates cells at the start of S phase. Seven hours after release from the second thymidine block, populations were used for nuclear preparations. At this point, a majority of the cells are in G2, but some laggards remain in S phase, and some rapidly cycling cells have entered mitosis and passed into Gl. To eliminate rapidly cycling cells, nocodazole (0.04 pg/ml) was added after release from the second thymidine block, to accumulate any cells entering mitosis in metaphase; such cells were largely shaken off from the GP-enriched population, and any remaining were clearly distinguishable from interphase cells. To distinguish slowly cycling cells still in S phase, cultures were given a 15 min pulse of 100 uM BrdU immediately before nuclear preparations were made. Preparation of HeLa Nuclei Intact nuclei with unpermeabilized nuclear membranes were prepared from Gl and G2 HeLa cells by treatment with the bacterial exotoxin SLO (Wellcome Diagnostics) or with the detergent digitonin (Calbiothem). Specifically, HeLa cells grown in culture dishes were washed threetimeswith PuckssalineAanddetached bytrypsinization. Phenylmethylsulfonyl fluoride(PMSF)(Sigma) was added to a final concentration of 1 mM, and the cells were pelleted by centrifugation at 500 rpm for 5 min in a H6000A rotor (Sorval). The intact cell pellet was resuspended in saline A containing PMSF, pelleted, and rinsed two times. For permeabilization of cells with SLO, the cell pellet was resuspended in ice-cold PIPES buffer (50 mM K-PIPES [pH 7.01, 50 mM KCI, 5 mM MgCl*, 2 mM EGTA) containing 1 uglml each aprotinin, leupeptin, and pepstatin (Sigma) and 1 mM dithiothreitol (Sigma), which was added fresh. Cell numbers were determined using a hemacytometer, and PIPES buffer was added to yield ~5 x IO5 cells per ml. An equal volume of ice-cold PIPES buffer containing SLO at a concentration of 1.5 IUlml was added giving a final concentration of SLO equal to 0.75 IUlml at 2.5 x IO5 cells per ml. Cells were held on Ice for lOto30min,and thetubeswereinvertedevery5min. Duringthis Incubation period, integrity of the plasma membrane was monitored by Incubation of cells with affinity-purified TRITC-IgG (Sigma). Routinely,

90% of cells showed cytoplasmic inclusion but nuclear exclusion of IgG were used in the studies described here. However, it should be noted that permeabilization of HeLa cells in some other preparations not used in these studies was extremely variable. We found that extending the incubation time, for cells at 0%’ in the presence of SLO. resulted in more uniform permeabilization. But even when cells were incubated with SLO for up to 30 min. uniform permeabilization was not achieved in some batches of cells. The reason for this variability is at present unclear. SLO-prepared nuclei were permeabilized with NP-40 (Pierce). Specifically, freshly thawed, unpermeabilized nuclei were pelleted and resuspended in PIPES buffer (ml x IO5 nuclei per ml), and an equal volumeof PIPES buffercontaining NP-40wasadded. Thefinalconcentration of NP-40 used was from 0.1% to 0.25%. Nuclei were incubated on ice, and nuclear permeability was monitored by incubation with TRITC-IgG. When >99% of nuclei showed a distinct uniform nuclear fluorescence, an equal volume of PIPES buffer containing 3% bovine serum albumin was added to prevent further permeabilization. The nuclei were pelleted and rinsed as described above and used immediately. For permeabilization of cells with digitonin, the unpermeabilized cell pellet described above was resuspended in transport buffer (Adam et al., 1990). and cell numbers were determined as described above. Transport buffer was added to yield ~1 x lo6 cells per ml. An equal volume of transport buffer containing 100 uglml digitonin was added, giving a final concentration of digitonin equal to 50 uglml at 5 x lo5 cells per ml. Cells were held on ice until >90% showed cytoplasmic inclusion but nuclear exclusion of TRITC-IgG. Nuclei were rinsed two times and finally resuspended in transport buffer. Digitonin-prepared nuclei were permeabilized with NP-40 in transport buffer as described for SLO nuclei above. SLO- and digitonm-prepared unpermeabilized nuclei were used fresh or were used freshly thawed following freezing in PIPES buffer or transport buffer supplemented with 5% dimethyl sulfoxide. Aliquots were frozen by placing tubes in foam racks at -20% for 30 min and then at -80%. Aliquots of nuclei were thawed rapidly by placing tubes in 23OC water. In most cases, one freeze/thaw cycle did not significantly alter the permeability of the nuclear membrane to TRITC-IgG. Only nuclei that showed little change in nuclear membrane permeability were used in additional experiments. The concentration of DNA in the final aliquots was estimated spectrophotometrically by measuring absorbance at 260 and 280 nm. In Vitro Replication Egg extract was thawed and supplemented wrth an energy regeneratmg system (Blow and Laskey, 1986) and cycloheximide as previously described (Leno and Laskey, 1991). HeLa nuclei were added at approximately 3 ng of DNA per ul of extract and labeled with 100 t&i/ml ]a-32P]dATP (800 Cilmmol. New England Nuclear) or 20 uM 5-biotin1 I-deoxyuridine triphosphate (biotin-1 I-dUTP, Sigma). Samples were incubated at 23°C for 6 hr. Determination of the extent of [a-32P]dATP incorporation and the processing of biotin-labeled nuclei for fluorescent microscopywasasdescribed previously(Leno and Laskey. 1991). To detect incorporated BrdU, EGS-fixed nuclei were spun onto coverslips and incubated in PES:4N HCI (1:l) for 30 min to denature the DNA. Nuclei were washed three times with Tween-Tns-buffered saline (TTBS) and subsequently incubated for 2 hr with anti-BrdU monoclonal antibody (Beckton Dickinson) in TTBS. Nuclei were washed two times with TTBS and incubated with Texas red anti-mouse immunoglobulin (Amersham). Density substitution was conducted as previously described (Leno

Cell 158

and Laskey, 1991). HeLa nuclei, prepared from cells that were not pulse-labeled with BrdU prior to nuclear isolation, were incubated at 3 ng of DNA per ul of extract with 0.25 mM BrdUTP (Sigma) and 100 uCi/ml [@P]dATP for 6 hr. Substituted DNA was separated by centrifugation to equilibrium (>40 hr at 20“) in a cesium chloride gradient. Fractions were collected, placed on GF-C filters (Whatman) containing 50 frg salmon sperm DNA as carrier, and the DNA precipitated with trichloroacetic acid. Free label was removed by washing filters and counts per minute determined by liquid scintillation.

We are grateful to Ft. T. Johnson for discussions about cell cycle controls, to Tony Mills for help with the confocal microscopy, and to Roger Northfield for skilled assistance. We also thank Joe Makkerh for information about streptolysin 0. This work was supported by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. October

Hennessy, localization 2252-2263.

K. M., Clark, C. D., and Botstein, D. (1990). Subcellular of yeast CDC46 varies with the cell cycle. Genes Dev. 4,

Hennessy, K. M., Lee, A., Chen, E., and Botstein, D. (1991). A group of interacting yeast DNA replication genes. Genes Dev. 5, 958-969. Hugo, F.. Reichwein, Use of a monoclonal brane pore formation

Acknowledgments

Received

Duncan, J. L., and Schlegel, Ft. (1975). Effect of streptolysin 0 on erythrocyte membranes, liposomes. and lipid dispersions. A proteincholesterol interaction. J. Cell Biol. 67, 160-173.

24, 1991; revised

January

15, 1992.

L. (1990). Nuclear protein import requires soluble cytoplasmic fac-

Ahnert-Hilger, G., Mach, W., Fohr, K. J., and Gratzl, M. (1989a). Poration by a-toxin and streptolysin-0: an approach to analyze intracellular processes. Meth. Cell Biol. 37, 63-90. Ahnert-Hilger, G., Bader, M. F., Bhakdi, S., and Gratzl, M. (1989b). Introduction of macromolecules into bovine adrenal medullaty chromaffin cells and rat pheochromocytoma cells (PC12) by permeabilization with streptolysin-0: inhibitory effect of tetanus toxin on catecholamine secretion. J. Neurochem. 52, 1751-1758. Alouf, J. E. (1980). Streptococcal erythrogenic toxin). Pharmacol.

toxins (streptolysin-0, Ther. 17, 661-717.

streptolysin-S,

Bittner, M. A., and Holz, R. W. (1988). Effects of tetanus catecholamine release from intact and digitonin-permeabilized maffin cells. J. Neurochem. 57, 451-456. Blow, J. J., and Laskey, R. A. (1986). nuclei and purified DNA by a cell-free 47, 577-587.

Jost, E., and Johnson, R. T. (1981). Nuclear laminaassembly, sis and disaggregation during the cell cycle in synchronized cells. J. Cell Sci. 47, 25-53.

syntheHeLa

Lazarovici, P., Fujita, K., Contreras, M. L., DiOrio, J. P., and Lelkes, P. I. (1989). Affinity purified tetanus toxin binds isolated chromaffin granules and inhibits catecholamine release in digitonin-permeabilized chromaffin cells. FEBS Lett. 253, 121-128. Leno, G. H., and Laskey, R. A. (1991). The nuclear membrane mines the timing of DNA replication in Xenopus egg extracts. Biol. 72, 557-566.

deterJ. Cell

Lohka, M. J., and Masui, Y. (1983). Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220, 719-721.

References Adam, S. A., Marr, R. S., andGerace, in permeabilized mammalian cells tors. J. Cell Biol. 17 1, 807-816.

J., Arvand, M., Kramer, S., and Bhakdi, S. (1986). antibody to determine the mode of transmemby streptolysin-0. Infect. Immunol. 54,641-645.

toxin on chro-

Initiation of DNA replication in extract of Xenopus eggs. Cell

Lohka, M. J., and Masui, Y. (1984). Roles of cytosol and cytoplasmic particles in nuclear membrane assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. J. Cell Biol. 98, 1222-l 230. Newport, J. (1987). around protein-free

Nuclear reconstitution in vitro: stages DNA. Cell 48, 205-217.

of assembly

Ojcius, D. M., and Young, J. D.-E. (1991). Cytolytic pore-forming teins and peptides: is there a common structural motif? Trends them. Sci. 76, 225-229. Paine, P. L. (1975). Nucleocytoplasmic ers microinjected into living salivary 657.

proBio-

movement of fluorescent tracgland cells. J. Cell Biol. 66, 652-

Peppers, S. C., and Holz. R. W. (1986). Catecholamine secretion from digitonin-treated PC12 cells, Effects of Cat+, ATP and protein kinase C activators. J. Biol. Chem. 267, 14665-14670. Prigent, sterols.

D., and Alouf, J. E. (1976). Interaction Biochim. Biophys. Acta 443, 288-300.

of streptolysin-0

with

Rao, P. N., and Johnson, R. T. (1970). Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature 225, 159-164.

Blow, J. J., and Laskey, R. A. (1988). A role for the nuclear membrane in controlling DNA replication within the cell cycle. Nature 332, 546548.

Sarafian, T., Aunis, D., and Eader, M.-F. (1987). Loss of proteins from digitonin-permeabilized adrenal chromaffin cells essential for exocytosis. J. Biol. Chem. 262, 16671-16676.

Blow, J. J., and Sleeman, A. M. (1990). Replication of purified DNA in Xenopus egg extract is dependent on nuclear assembly. J. Cell Sci. 95, 383-391.

Sheehan, M. A., Mills, A. D., Sleeman, A. M., Laskey, R. A., and Blow, J. J. (1988). Steps in the assembly of replication competent nuclei in a cell-free system from Xenopus eggs. J. Cell Biol. 706, 1-12.

Blow, J. J., and Watson J. V. (1987). Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system. EMBO J. 6, 1997-2002.

Wilson, S. P., and Kirshner, N. (1983). Calcium evoked digitonin-permeabilized adrenal medullary chromaffin Chem. 258, 4994-5000.

Brown, D. B., Hanks, S. K., Murphy, E. C., and Rao, P. N. (1985). Early initiation of DNA synthesis in Gl phase HeLa cells following fusion with red cell ghosts loaded with S phase cell extracts. Exp. Cell Res. 756,251-259.

Yan, H., Gibson, S., and Tye, B. K. (1991). Mcm2 and Mcm3. two proteins important for ARS activity, are related in structure and function. Genes Dev. 5. 944-957.

De Roeper, A., Smith, J. A., Watt, R. A., and Barry, J. M. (1977). Chromatin dispersal and DNA synthesis in Gl and G2 HeLa cell nuclei injected in Xenopus eggs. Nature 265. 469-470. Downes, C. S., and Collins, A. R. S. (1982). Effects of DNA replication inhibitors on UV excision repair in synchronized human cells. Nucl. Acids Res. 70. 5357-5368. Downes, C. S.. Collins, damage in synchronized J. 25, 129-150.

A. R. S., and Johnson, R. T. (1979). DNA HeLa cells irradiated with ultraviolet. Biophys.

Downes, C. S., Unwin, D. M., Northfield, R. G. W., and Berry, M. J. (1987). Automatic nitrous oxide synchronization of mitotic human cell cultures. Anal. Biochem. 765, 56-58.

secretion from cells. J. Biol.