Efficient Plasmid DNA Replication in Xenopus Egg Extracts Does Not Depend on Prior Chromatin Assembly

doi:10.1074/jbc.270.50.29676

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Volume 270, Number 50, Issue of December 15, 1995 pp. 29676-29681
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient Plasmid DNA Replication in Xenopus Egg Extracts Does Not Depend on Prior Chromatin Assembly (*)

(Received for publication, August 21, 1995; and in revised form, September 25, 1995)

J. Aquiles Sanchez (§), , Diane R. Wonsey , Leia Harris , Joanella Morales , Lawrence J. Wangh

From the Department of Biology, Brandeis University, Waltham, Massachusetts 02254

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

ACKNOWLEDGEMENTS

REFERENCES

ABSTRACT

Small plasmids replicate efficiently in unfertilized Xenopus eggs provided they are injected before rather than after activation of the cell cycle. Here we use Xenopus egg extracts to test the hypothesis that efficient replication results from chromatin assembly prior to activation giving preloaded plasmids a head start toward the formation of a replicating pseudonucleus (Sanchez, J. A., Marek, D., and Wangh, L. J.(1992) J. Cell Sci. 103, 907-918). As in ovum, plasmid DNA preincubated in unactivated egg cytoplasmcytostatic factor extracts) replicate more efficiently after extract activation than does the same DNA added to the same extract after activation. Unlike in ovum, however, plasmids that replicate efficiently in vitro do not assemble into chromatin during preincubation and become topologically knotted instead. But even DNA knotting does not explain subsequent efficient replication. Also, plasmids preassembled into chromatin in vitro do not replicate efficiently in activated egg cytoplasm unless first preincubated in a CSF extract. We conclude that unactivated eggs contain replication-enhancing activities that can act independently of plasmid chromatin assembly and DNA topology. These postulated ``preloading'' factor(s) may be related to licensing factor, an activity that controls initiation of DNA replication in eukaryotic cells. The experimental conditions described here will permit characterization of preloading/licensing factor(s) in the context of a small plasmid substrate.

INTRODUCTION

Chromatin assembly plays a central role in the pathway of nuclear formation leading to plasmid DNA replication in Xenopus eggs and egg extracts(1) . Newport (2) first showed that plasmids assembled into chromatin bind nuclear membrane vesicles that fuse to form a nuclear envelope, complete with pores and lamina. The resulting pseudonuclei are essential for initiation of plasmid DNA replication (3, 4) .

Our previous studies also pointed to early chromatin assembly as an important step leading to plasmid replication in intact eggs. We have shown that plasmids replicate efficiently in activated eggs, provided they are injected before rather than after the start of the first cell cycle(5) . We have called this phenomenon the preloading effect and have correlated both the amount and the timing of plasmid replication to the extent of chromatin assembly prior to activation(6) . We have suggested that chromatin assembly before the start of the cell cycle leads to efficient replication after activation because it gives molecules a head start toward the formation of pseudonuclei.

But there may be other reasons why incubation of a plasmid in the cytoplasm of an egg arrested in meiotic metaphase II leads to efficient replication. For instance, these eggs may contain enzymatic activities or DNA binding proteins other than histones that can directly enhance replication initiation after activation. This possibility is in accord with our observation that plasmid molecules injected into eggs that have already entered the first cell cycle show increased replication during the second cell cycle (i.e. after passage through the first mitosis)(6) . ()It also fits the ``licensing factor hypothesis'' put forward by Blow and Laskey (7) . These investigators argue that eukaryotic nuclei normally replicate only once per cell cycle because they have to pass through mitosis, or at least experience nuclear envelope breakdown, before they can initiate DNA synthesis a second time.

Given these alternative possibilities, we decided to directly determine whether prior chromatin assembly actually accounts for efficient plasmid replication after the start of the cell cycle. In order to facilitate this analysis we first identified conditions for efficient plasmid DNA replication in vitro. We report here that efficient in vitro replication, like efficient in ovum replication, depends on exposure of plasmid to cytoplasm of unactivated Xenopus eggs (CSF ()extracts). But contrary to our original hypothesis, chromatin assembly in this cytoplasm is not required for subsequent efficient plasmid replication. This observation was confirmed by attempting to replicate preassembled plasmid chromatin directly in activated egg extracts. Once again we observed that exposure of the template to the unactivated egg cytoplasm is required for subsequent efficient replication, regardless of the initial degree of plasmid chromatin assembly.

In the course of this investigation, we also discovered that CSF extracts cause closed circular plasmid molecules to become topologically knotted. This observation led us to examine whether DNA knotting in unactivated egg cytoplasm might account for efficient in vitro replication. Our results establish that neither chromatin assembly nor DNA knotting before the start of the cell cycle accounts for subsequent efficient plasmid replication. We conclude that the preloading effect is most likely due to additional specific DNA-protein interactions in unactivated egg cytoplasm. A possible candidate for these preloading factor(s) is replication licensing factor, an activity responsible for cell cycle regulation of DNA replication whose components have started to be identified recently (8, 9, 10) . The in vitro system described here will permit characterization of this and other replication-enhancing activities of the mitotic egg cytoplasm in the context of an easily characterized plasmid substrate instead of the complex genome of whole eukaryotic nuclei.

MATERIALS AND METHODS

In Vivo Analysis of Plasmid DNA Replication

In vivo analysis of FV1 DNA replication and chromatin was carried out as described previously (6) .

Extract Preparation

Low speed extracts from metaphase arrested eggs (CSF extracts) were prepared according to the protocol of Wangh et al.(11) and were used fresh rather than frozen and thawed. Extracts were supplemented with a final concentration of 10 mM creatine phosphate, 10 µg/ml creatine kinase, and 0.1 mM CaCl (2)

. When specified, beta

-glycerol-PO (4)

was added to a final concentration of 80 mM. At the times indicated in the text, the CSF extract was induced to enter the cell cycle by the addition of calcium to a final concentration of 1.2 mM CaCl (2)

as measured by a precipitous decline in H1 kinase activity. Activation with 3 mM CaCl (2)

as recommended by Blow and Sleeman (3) was found to impede progress of the cell cycle in the extract (data not shown).

To prepare high speed extracts from activated eggs, eggs were activated for 28 min at 20 °C and then processed as described by Wangh et al.(11) . The resulting low speed supernatant was further centrifuged in a SW50.1 rotor for 60 min at 45,000 rpm. The clear cytoplasmic layer was spun once more at 45,000 rpm for 30 min, adjusted to 7.5% glycerol (v/v), and then frozen in 20-µl aliquots in liquid nitrogen.

DNA Isolation from Egg Extracts

In a typical experiment, 2-10-µl aliquots of extract containing FV1 plasmid DNA were rapidly frozen on dry ice and were subsequently thawed and thoroughly dissolved by the addition of an equal volume of GuHCl buffer (4.5 M guanidine-HCl, 0.1 M EDTA, pH 8.0, 0.15 M NaCl, and 0.05% sarkosyl) (5, 6) . The salt concentration was then decreased by addition of 150 µl of STE buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, and 50 mM EDTA, pH 8.0) supplemented with 20 µg of tRNA as a carrier, followed by the addition of 200 µl of proteinase K Buffer (1 times

STE, 1% sarkosyl, and 1 mg/ml proteinase K). After incubation at 37-50 °C for 1-2 h, the samples were extracted twice with phenol:chloroform, followed by ethanol precipitation and resuspension in TE/RNase buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0, and 100 µg/ml RNase A).

For isolation of non-nicked knotted plasmid molecules, the above protocol was modified to allow for closure of topoisomerase II-dependent nicks and double strand breaks(12) . Aliquots of extract were adjusted to 0.5 M NaCl and were incubated at room temperature for 30 min prior to freezing on dry ice. Subsequent DNA isolation steps were carried out as described above.

Analysis of DNA Replication and Chromatin Assembly

FV1 replication and chromatin assembly were measured by the DpnI resistance assay and the supercoiling assay as described previously(6) . Southern hybridization signals were quantitated using a PhosphorImager (Molecular Dynamics), and replication efficiencies were calculated as the ratio of DpnI-resistant (replicated) material to the total amount of DNA/sample.

Generation, Assessment, and Replication of Chromatin Templates

FV1 DNA was added to a frozen and thawed high speed activated extract at a concentration of 16 ng/µl, and multiple aliquots were collected at regular intervals and frozen immediately on dry ice. The DNA from one aliquot at each time was purified and used to assess the level of chromatin assembly by examining the appearance of negatively supercoiled DNA in agarose gels containing 20 µg/ml chloroquine(6) . In order to determine how efficiently each chromatin sample replicated, another aliquot was thawed and diluted 1:8 into a freshly prepared CSF extract either 10 min before or 10 min after activation of the extract by addition of calcium.

Characterization of Nicked Knotted and Knotted DNA Molecules

Nicked knotted DNA molecules migrate very differently from negatively supercoiled molecules on one- and two-dimensional chloroquine gels(13, 14, 15) . One-dimensional chloroquine gels were carried out as described before(6) . For two-dimensional chloroquine gel electrophoresis(16) , 1 ng of knotted DNA was mixed with 1 ng of a collection of all possible FV1 negatively supercoiled topoisomers generated according to Bowater et al.(17) . This DNA mixture was fractionated in the first dimension in a 0.72% agarose gel in 1 times

TPE (50 mM Tris, 10 mM EDTA, pH 7.2, with 85% phosphoric acid) buffer containing 0.75 µg/ml chloroquine at 70 V for 18 h. The gel was then equilibrated in 1 times

TPE buffer supplemented with 4.5 µg/ml chloroquine for 7 h and was run in the orthogonal dimension at 70 V for 16 h. Buffer was recirculated in both the first and second dimensions. Fractionated molecules were visualized via Southern transfer, hybridization, and autoradiography as described before(6) .

RESULTS

The Preloading Effect in Ovum

Injection of plasmid DNA into unactivated eggs prior to the start of the cell cycle dramatically increases the efficiency of replication after cell cycle activation. FV1 molecules injected into unactivated Xenopus eggs do not replicate and therefore remain DpnI-sensitive (Fig. 1, lane A). But a high percentage of these molecules replicates during the first S phase after egg activation. As a result, full-length DpnI-resistant molecules accumulate and DpnI-sensitive molecules decrease (Fig. 1, lane B). In contrast, very few FV1 molecules injected directly into activated eggs replicate during the first S phase (Fig. 1, lane C), although a significant percentage of these molecules do replicate in the second S phase (data not shown, but see (6) )

Figure 1: The preloading effect in ovum.Left panel, efficient replication of preloaded DNA. Unfertilized Xenopus eggs were injected with 1 ng of FV1 DNA before (lanes A and B) or after (lane C) activation by calcium ionophore treatment. FV1 DNA was then recovered and digested with DpnI to determine the absence or the presence of replicated molecules(6) . Lane A, plasmid recovered immediately before activation, prior to the start of the cell cycle. Lanes B and C, plasmids recovered 70 min after activation. Notice that no FV1 replication occurred before activation and that only plasmids injected prior to the start of the cell cycle replicated efficiently after activation. Right panel, plasmids preincubated in unactivated eggs are more assembled into chromatin by the beginning of the S phase. Eggs were injected with 1 ng of FV1 DNA before (lanes D and E) or after (lane F) activation by calcium ionophore treatment. FV1 DNA was then recovered before (lane D) or 30 min after activation (lanes E and F) and analyzed by agarose gel electrophoresis in the presence of 18 µg/ml chloroquine. Under these conditions, form IIr (relaxed) closed circles move most rapidly, and form I molecules containing increasing numbers of supercoils (one supercoil/nucleosome incorporated in ovum) migrate with decreasing mobility. Plasmids preloaded into unactivated eggs assemble into chromatin before activation and consequently have more nucleosomes at the start of the first S phase than nonpreloaded plasmids. The additional band (arrow, lane D) is nicked knotted DNA (see text).

Why do plasmids preloaded in unactivated eggs replicate efficiently after activation? Once in the unactivated egg, negatively supercoiled molecules first relax into closed circles and then assemble nucleosomes. When the DNA is purified, the process of chromatin assembly is observed as a gradual upward shift in a ladder of negatively supercoiled topoisomers resolved on a chloroquine agarose gel (one negative supercoil for each nucleosome) (Fig. 1, lane D). When the cytoplasm enters the S phase of the first cell cycle about 27 min after calcium ionophore activation(11) , preloaded FV1 molecules have more nucleosomes than do FV1 molecules injected into eggs 10 min after activation (Fig. 1, lanes E and F). Observations like this led us to postulate that preloaded FV1 molecules replicate more efficiently because they have a head start on the pathway leading to replication in the first cell cycle(6) .

The Preloading Effect in Vitro

Preincubation of FV1 DNA in a CSF extract prepared from unactivated eggs also results in efficient plasmid replication once the extract is activated by addition of CaCl (2)

(Table 1). 2 h of preincubation in CSF extracts results in replication of 30-40% of the input FV1 DNA, an efficiency comparable to that observed in ovum(6) . However, the kinetics of in vitro replication are somewhat slower (Fig. 2). In vitro replication starts 60-90 min after the addition of calcium and reaches a maximum (32% in this experiment) by 240 min. In contrast, only a small percentage (5%) of FV1 molecules replicates when the DNA is added to the CSF extract 10 min after the addition of calcium. Twice replicated molecules are not recovered in either case in the in vitro system, and measurements of histone H1 kinase activity (data not shown) demonstrate that mitosis does not take place in these activated egg extracts. We conclude that in this in vitro system, as in intact eggs, exposure of FV1 DNA to unactivated egg cytoplasm enhances subsequent plasmid replication.

Figure 2: The preloading effect in vitro: kinetics of FV1 DNA replication. FV1 DNA (final concentration, 4 ng/µl) was preincubated for 120 min in a freshly prepared CSF extract. The extract was then activated and sampled over time for replicated molecules. An additional aliquot of FV1 DNA was added directly to a CSF extract 10 min after activation and was assayed for replication. The results show that preincubated DNA replicates efficiently and over a long period of time, whereas DNA that is not preincubated hardly replicates at all. The percentage of replicated molecules corresponds to the fraction of each sample that is resistant to digestion with DpnI.

Table 1also shows that only a very brief exposure to the unactivated egg extract is required to significantly increase the efficiency of replication. For instance, preincubation in CSF extract for as little as 1 min is sufficient to increase FV1 replication efficiency 3-fold (19 versus 6% replication for nonpreincubated DNA) (see Table 1). However, little if any chromatin could have assembled in such a short preincubation time. This observation led us to question whether chromatin assembly in CSF extracts is, in fact, required for efficient replication.

Chromatin Assembly and the Preloading Effect in Vitro

Further doubts about the importance of prior chromatin assembly came from chloroquine gel analysis of FV1 molecules that had and had not been incubated in CSF extract for 2 h before activation of the cell cycle. Because preincubated molecules replicated much more efficiently (Fig. 2), we expected them to be more highly supercoiled at the very start of the in vitro cell cycle, as observed in eggs (Fig. 1, right panel). However, this was not the case. In fact, both preincubated and nonpreincubated plasmids display the same level of chromatin assembly at equivalent times after activation (data not shown, but see Fig. 4A). We conclude that chromatin assembly does not take place prior to the addition of calcium and therefore cannot account for efficient plasmid replication after activation.

Figure 4: Plasmids recovered from CSF extracts exhibit a new, unusual topological conformation. A, one-dimensional chloroquine gel analysis. FV1 DNA (final concentration, 4 ng/µl) was preincubated in a CSF extract for up to 120 min. At this time the extract was activated with calcium and incubated for an additional 240 min. Plasmid DNA recovered at different time points throughout the experiment was analyzed by chloroquine agarose gel electrophoresis. Lane C, input supercoiled DNA. Lane T, relaxed closed circular FV1 DNA. Negative numbers, time (in min) after DNA addition to the CSF extract. Positive numbers, time (in min) after extract activation. FV1 recovered from CSF extracts display topoisomeric ladders with rungs whose intensity and spacing differs from that of FV1 ladders recovered after activation. The white arrow indicates the most abundant of these new rungs, which disappears between 30 and 75 min after activation. The lower strong band in this ladder, which persists after activation, corresponds to form III linear molecules. The figure is the negative image of an autoradiogram. B, two-dimensional chloroquine gel analysis. Two-dimensional chloroquine gel analysis of a mixture of FV1 DNA recovered from CSF extracts (t = 0 sample in (A) and a standard set of negatively supercoiled FV1 topoisomers. NC indicates nicked circular molecules. The bracket indicates the positions of nicked trefoil and complex knotted DNA molecules. Molecules migrating in a line under the nicked knotted molecules correspond to minor degradation products of linear DNA (L).

Replication of Plasmid Chromatin Templates in Vitro

The above conclusion was independently confirmed by examining the replication of preassembled chromatin templates in vitro. In this experiment, we prepared FV1 chromatin with increasing numbers of nucleosomes/molecule by incubating the plasmid in a high speed supernatant prepared from activated eggs (18) for up to 570 min (Fig. 3A). DpnI digestion of each of these DNAs (data not shown) revealed that none had replicated. This was expected because the high speed activated extract lacks the membrane vesicles required for pseudonucleus formation(3, 19, 20) . Next, we diluted each of the preassembled chromatin templates into a CSF extract either ten minutes before or immediately after addition of calcium. According to our original hypothesis, we expected preassembled chromatin templates to replicate efficiently in activated extracts, without need of any additional exposure to the unactivated CSF cytoplasm. This was not the case.

Figure 3: Replication of preassembled chromatin templates in vitro.A, assembly of plasmid chromatin templates in high speed activated extracts. FV1 DNA was added to a frozen and thawed high speed activated extract to a final concentration of 16 ng/µl. At the times indicated aliquots were taken and frozen on solid CO (2) . Chloroquine gel analysis reveals that chromatin assembly increases as a function of incubation time and is complete by 570 min. Longer incubations (data not shown) did not further increase chromatin assembly. B, chromatin templates still require CSF preincubation to replicate efficiently. Frozen and thawed aliquots containing either naked DNA or highly assembled chromatin in high speed activated egg extract (0 min sample and 570 min sample shown in Fig. 4A, respectively) were diluted into a CSF extract 10 min before (right panel) or after (left panel) calcium addition. FV1 DNA was recovered at the indicated times and was assayed for DpnI-resistant DNA in each sample. The results show that independently of the amount of chromatin assembly only those templates preincubated in the CSF extract replicated efficiently.

Even the template assembled into chromatin for 570 min replicated poorly when added directly to the calcium-treated CSF extract (Fig. 3B). The same was also true for templates with fewer nucleosomes/molecule (data not shown). In contrast, incubation of these same chromatin templates in unactivated CSF extract for 10 min prior to addition of calcium resulted in efficient replication (Fig. 3B). These results clearly demonstrate that prior chromatin assembly per se does not account for the preloading effect.

FV1 Becomes Topologically Knotted in CSF Extracts

Our results suggest that naked FV1 DNA molecules added directly to a CSF extract do not assemble nucleosomes before the start of the cell cycle. Instead these molecules are converted to a new unusual topological form that migrates on a one-dimensional chloroquine gel as a ladder of widely spaced bands descending from the position of form II, nicked circles (Fig. 4A). This ladder is distinct from that of negatively supercoiled topoisomers whose rungs are more closely spaced and migrate at different distances from the position of Form II DNA depending on their degree of supercoiling (Fig. 4A). FV1 molecules adopt their unusual conformation as soon as they are added to the CSF extract and remain in this conformation for 30-45 min after extract activation by addition of calcium (Fig. 4A and data not shown). The most abundant of these unusual bands is also formed in ovum, albeit as a much smaller fraction of the total sample (see Fig. 1, lane D). Given these facts, we sought to identify this unique FV1 conformation and to investigate its possible contribution to the preloading effect.

Two-dimensional chloroquine gel analysis (16) established that the new forms of FV1 recovered from CSF extracts correspond to nicked knotted plasmids (Fig. 4B). Whereas a standard set of negatively supercoiled molecules forms an arc of discrete spots in these two-dimensional gels, the ladder of FV1 molecules recovered from CSF extracts migrates in a straight diagonal line, i.e. independently of chloroquine concentration. This pattern of migration is characteristic of a family of nicked knotted DNA circles formed when plasmid DNA is exposed to high concentrations of topoisomerase II in vitro(13, 14, 15) . The most prominent band below the nick circles is the trefoil or pretzel form and has only a single knot, whereas the more rapidly migrating but less abundant forms have increasing numbers of knots(13) . DNA knotting in egg extracts is probably due to the large stockpiles of topoisomerase II present in Xenopus eggs(21) .

The experiments shown in Fig. 5confirmed that plasmid DNA recovered from CSF extracts is indeed knotted. DNA knotting requires a high ratio of active topoisomerase II to plasmid DNA molecules(12, 13, 14) . Accordingly, increasing plasmid DNA concentration in the CSF extract or lowering topoisomerase II activity by supplementing the CSF extract with the phosphatase inhibitor beta -glycerol-PO (4) inhibited DNA knotting to different extents (Fig. 5). Interestingly, the same conditions that prevent DNA knotting also allow generation of a ladder of FV1 topoisomers characteristic of chromatin templates (Fig. 5). We conclude that rapid plasmid DNA knotting hinders chromatin assembly in the CSF extract.

Figure 5: Altering the ratio of plasmid DNA to active topoisomerase II in CSF extracts affects DNA knotting. The ratio of plasmid DNA to active topoisomerase II in the CSF extract was altered by either increasing the amount of plasmid DNA or by supplementing the extract with beta -glycerol-PO (4) , which decreases topoisomerase II activity. For this experiment, FV1 DNA was added to a CSF extract at either 1.5 or 16 ng/µl in the presence or the absence of 80 mM beta -glycerol-PO (4) . Aliquots of the extract were collected at regular intervals thereafter and were analyzed for plasmid topology by chloroquine gel electrophoresis. C, input plasmid DNA; T, FV1 DNA relaxed by topoisomerase I; L, linear FV1 DNA.

DNA Knotting in CSF Extracts Does Not Account for the Preloading Effect in Vitro

Although extensive, DNA knotting observed in unactivated CSF extracts is unlikely to account for efficient plasmid replication after activation, because high levels of DNA knotting are not observed in unactivated whole eggs injected with FV1 (see Fig. 1, lane D). Nevertheless, we could not rule out a role for knotting in vitro because beta

-glycerol-P0 (4)

, which suppresses knotting in vitro (Fig. 5), also inhibits DNA synthesis (data not shown).

In order to make sure that knotting does not play role in the preloading effect, we directly compared the replication of purified, non-nicked knotted and supercoiled FV1 molecules added directly to freshly activated CSF extracts. As shown in Fig. 6A, nicking of knotted plasmids was prevented by addition of 0.5 M NaCl to samples prior to DNA isolation (see (14) and ``Materials and Methods''). Both knotted and supercoiled molecules replicated poorly in activated CSF extract (Fig. 6B). In contrast, control samples of supercoiled FV1 DNA added to CSF extracts before activation once again replicated efficiently, proving that the extract was fully competent for replication. We conclude that knotting per se does not promote efficient plasmid replication and consequently does not account for the preloading effect.

Figure 6: DNA knotting in CSF extracts does not account for the preloading effect in vitro. A, the addition of NaCl to CSF extracts prior to plasmid isolation prevents topoisomerase-mediated DNA nicking. Salt (final concentration, 0.5 M) was added 30 min prior to DNA isolation (+ and - indicate treated and untreated samples, respectively). Plasmids from salt-treated extracts migrate fast in chloroquine gels due to their very compact shape caused by DNA knotting. B, comparison of the extent of replication of purified intact knotted and supercoiled FV1 DNA added directly to freshly activated CSF extracts. Supercoiled FV1 DNA or intact knotted FV1 DNA (a separate aliquot of the sample shown in A) were added directly to an activated CSF extract (final concentration, 4 ng/µl) and analyzed for replication 60 and 240 min later. As a control for the competency of the extract, supercoiled FV1 DNA was preincubated in the same CSF extract for the times indicated in the figure and then assayed for replication 60 and 240 min after extract activation by calcium addition. Intact knotted DNA did not replicate efficiently in the activated CSF extract.

DISCUSSION

This paper describes a new experimental system for the efficient replication of small circular plasmid DNAs in extracts prepared from unfertilized Xenopus eggs. Incubation of plasmid molecules in metaphase arrested extracts, like unactivated whole eggs, enhances their subsequent replication when the cytoplasm or intact egg is activated to re-enter the cell cycle. In contrast, the same DNA added directly to an already activated cytoplasm, or egg, replicates poorly. Efficient plasmid replication takes place in freshly prepared CSF extracts but has thus far failed in frozen and thawed CSF extracts activated by addition of calcium. This is probably because unlike fresh extracts, frozen and thawed CSF extracts can only be fully activated by diluting them into a second extract prepared from activated eggs (11) .

Even though our in vitro system duplicates the preloading effect, our analysis of chromatin assembly in vitro does not support our earlier conclusion from intact eggs that efficient replication depends on chromatin assembly before the start of the cell cycle(6) . For instance, although FV1 is rapidly knotted in CSF extract and does not assemble into chromatin, it nevertheless replicates efficiently after activation. Conversely, preassembled chromatin templates do not replicate efficiently in activated egg extracts unless these templates are briefly exposed to an unactivated CSF extract. We conclude that efficient replication depends on factors or enzymatic activities present in CSF extract that are distinct from those required for chromatin assembly or knotting.

In view of the findings reported here, how can we explain our results using intact eggs, in which both the timing and the amount of replication appeared to quantitatively correlate with the extent of prior chromatin assembly(6) ? It has been clearly established that plasmid replication in Xenopus eggs and extracts requires previous chromatin assembly, formation of a pseudonucleus, and assembly and activation of DNA replication centers(2, 3) . Thus, the correlation between chromatin assembly and efficient replication in ovum is probably not fortuitous. However, chromatin assembly in unactivated eggs may mask the fact that other factors of the metaphase cytoplasm also bind to FV1 DNA and play a critical role in subsequent template replication.

In the course of these studies we also discovered that CSF extracts can introduce topological knots into plasmid DNA due to the high levels of topoisomerase II activity in these extracts. DNA knotting appears to explain the failure of CSF extracts to assemble plasmid chromatin, but we cannot rule out the possibility that additional replication-enhancing proteins bind to knotted FV1 DNA. Our findings demonstrate that CSF extracts prepared under standard conditions differ in important respects from the cytoplasm of intact unactivated eggs. Conditions that prevent DNA knotting in vitro favor chromatin assembly, which predominates in ovum. The fact that beta -glycerol-PO (4) , a phosphatase inhibitor, enhances chromatin assembly in vitro suggests that the ratio of kinases to phosphatase is higher in intact eggs than in extracts prepared under standard conditions. The effect is probably indirect, via regulation of topoisomerase II activity in the extract. Hyperphosphorylation of topoisomerase II is known to decrease its affinity for DNA(22) .

Finally, the fact that CSF extract enhances replication of preassembled chromatin makes it possible for the first time to distinguish between chromatin assembly per se and the biochemical changes in template structure required for replication. We now predict that the CSF determinants responsible for enhancing subsequent replication must 1) interact with both naked DNA and with DNA already assembled into chromatin, 2) bind to these substrates very rapidly, 3) disappear quickly from CSF extract upon addition of calcium, and 4) reappear in activated eggs when they progress into first mitosis.

The above characteristics of our replication-enhancing determinant are compatible with ``replication licensing factor'', an activity controlling initiation of nuclear DNA replication in Xenopus egg extracts(7) . Replication licensing factor is thought to gain access to the DNA during mitosis when nuclear envelope breakdown occurs and is believed to become active upon exit from metaphase(23, 24, 25) . In this regard, the preloading effect could be viewed as the result of mitotic egg cytoplasm licensing plasmid DNA for efficient replication. Accordingly, the Xenopus homologues of the yeast MCM3 and mammalian P1 family recently identified as components of licensing factor in frog egg extracts (8, 9, 10) become likely candidates for the preloading factor. We are currently investigating the relationship between licensing factor and preloading factor by testing the replication efficiency of plasmid DNA assembled into chromatin in vitro in the presence or the absence of MCM-3 protein in activated extracts devoid of licensing factor activity(23) . The in vitro system and experimental conditions described here will permit characterization of replication-enhancing activities in mitotic egg cytoplasm in the context of a small, well characterized plasmid substrate rather than in the context of the complex genome of whole eukaryotic nuclei.

FOOTNOTES

*: This work was supported by Grant 3206 from the Council for Tobacco Research (to L. J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§: To whom correspondence should be addressed. Tel.: 617-736-3111; Fax: 617-736-3107; Sanchez@binah.cc.brandeis.edu.
(): A. Aguilar and L. J. Wangh, unpublished observations.
(): The abbreviation used is: CSF, cytostatic factor.

ACKNOWLEDGEMENTS

We thank Dr. James Wang for his insights into DNA knotting.

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