INTRODUCTION

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Replication of the eukaryotic genome occurs exclusively during S phase and requires strict coordination among cis-acting sequences, the replicative enzymes, and their regulatory factors to ensure that origins of replication fire only once per cell cycle. The presence of multiple origins of replication on eukaryotic chromosomes permits the simultaneous engagement of polymerases at different sites on the same chromosome and also allows for delayed or regulated origin firing (25). Both the cis-acting sequences and the trans-acting factors that function at origins in the budding yeast Saccharomyces cerevisiae have been well characterized genetically, although no successful reconstitution of origin-dependent replication in a fully soluble system has been reported (6, 54).

All known yeast origins of replication include an essential 11-bp sequence, called the autonomously replicating sequence (ARS) consensus, situated within an AT-rich stretch of DNA that binds the nuclear scaffold (reviewed in reference 50). These elements confer high-frequency transformation on circular plasmids in S. cerevisiae (32, 63) and function as origins of DNA replication on both circular and linear plasmids and in the genome (7, 33). ARS elements occur on average once per 36 kb in yeast chromosomes (49), although not all are active origins in their native genomic context (50). Under certain conditions, a large fragment of yeast chromosome III appears capable of semiconservative replication in the absence of detectable initiation events at known ARS elements (48a). This fact suggests that alternative means to initiate replication may exist in vivo, although the mechanisms remain to be characterized.

The standard ARS-specific initiation of replication in yeast requires ORC, a six-subunit origin recognition complex (4, 16). Strains with temperature-sensitive alleles of ORC2 and ORC5 arrest in S phase with the dumbbell phenotype typical of mutants that affect DNA replication and show a drop in the efficiency of initiation (3, 40, 42). Moreover, G₁-phase nuclei isolated from a temperature-sensitive orc2-1 mutant fail to initiate DNA replication in vitro at the restrictive temperature (53). Although ORC is bound to the ARS consensus throughout the cell cycle (17), in vivo footprinting data indicate that additional components associate with ORC following entry into G₁ phase, forming the so-called prereplicative complex (pre-RC) (17). The transition from the postreplicative complex to the pre-RC coincides with the synthesis of Cdc6p (9, 55), a protein that is essential for the initiation of DNA replication and that associates with ORC (39, 40). Cdc6p, in turn, promotes the association of minichromosome maintenance (MCM) proteins with prereplicative chromatin (10, 21, 64). Activation through an S-phase-promoting factor is proposed to modify and possibly displace Cdc6p and the MCM proteins from the pre-RC, triggering the initiation of DNA replication (15, 47).

The initiation of DNA synthesis itself requires the catalytic activity of the polymerase  (pol )-primase complex, which is composed of four subunits, two of which (p48 and p58) are involved in short RNA primer synthesis on the template strand (reviewed in reference 27). The catalytic p180 subunit then extends the cDNA strand for 100 to 200 nucleotides. The exact function of the second largest subunit, p86, is unknown, but this subunit appears to be regulatory and may target the complex to prereplicative foci. In the well-characterized simian virus 40 (SV40) replication reaction, the pol -primase complex is targeted to the site of initiation through contacts with the single-strand-binding protein replication protein A (RPA) and the virus-encoded, origin-binding factor, large T antigen (11, 22, 41). RPA is also essential for early steps in genomic replication in eukaryotes, but it associates tightly with DNA near the origin only after activation of the Clb-Cdk1 kinase (65). The fact that replication-associated RPA foci form independently of ORC in nuclei reconstituted in Xenopus extracts (10) suggests that replication enzymes and factors may associate by a mechanism partially independent of the ORC-containing pre-RC. Consistently, a proliferating cell nuclear antigen (PCNA)-binding sequence motif that targets enzymes to replication foci in mammalian cells has been identified (8, 45).

In yeast, the protein kinase encoded by CDC28 (also called Cdk1) is a key regulator of the cell division cycle and, when complexed with B-type cyclins, this kinase is essential for promotion of S phase (reviewed in reference 48). The six B-type cyclins that stimulate Cdk1 are expressed at different points in the cell cycle. CLB5 and CLB6 expression peaks in early S phase, and although deletion of these genes delays the onset of S phase, other B-type cyclins can promote S phase if induced after the pre-RC has formed (2, 60). Not only does activation of the Clb-Cdc28 complex promote the G₁/S transition, but also the sustained activity of this complex through G₂ phase appears necessary to prevent the precocious reassembly of the pre-RC. Thus, the maintenance of high Clb-Cdk1 activity also ensures that origins fire only once per cell cycle (12). Cdc6p appears to be a physiological substrate of Clb-Cdc28, but other replication-associated targets undoubtedly exist, since mutation of the phosphoacceptor sites in Cdc6p has little effect on cell cycle progression (7a). Moreover, Clb-Cdc28 activity is a prerequisite for the association of Cdc45p with the pre-RC (69).

A second Ser/Thr kinase, encoded by CDC7, is also necessary for the initiation of DNA replication in vivo (5, 19, 31, 34) and in isolated yeast nuclei (54a). The genetically determined CDC7 execution point is downstream of the point of G₁/S function of CDC28 (31), and its activity is necessary throughout S phase to allow the activation of late-firing origins (5, 20). Mcm2p is at least one of the physiological substrates of the Dbf4-Cdc7 complex (38). Intriguingly, the requirement for both Cdc7p and its regulatory subunit, Dbf4p, can be bypassed by an allele of MCM5 called bob1-1 (30).

In vertebrate cells, promotion of S phase requires Cdk2 in complex with cyclins D1, E, and A (29, 51, 52, 57). Consistently, the promotion of replication in isolated G₁-phase HeLa cell nuclei requires the synergistic activity of CycA-Cdk2 and CycE-Cdk2 (37). The mitotic CycB-Cdc2 kinase does not stimulate replication in this mammalian in vitro replication system, yet in a similar reconstituted yeast system in which G₁-phase nuclear templates replicate semiconservatively (53), either the recombinant Xenopus CycB-Cdc2 kinase or the yeast Clb5-Cdc28 complex can serve to promote ORC-dependent initiation in vitro (54a). To further examine the ORC and kinase dependence of DNA replication in yeast, we have made use of a recently established soluble DNA replication assay in which supercoiled plasmids can be semiconservatively replicated in S-phase nuclear extracts. This reaction requires polymerase  (pol ), the pol -primase complex, and the Dna2 helicase (6). Both ribonucleotides and deoxynucleotides are required for successful replication, which is sensitive to aphidicolin but not -amanitin. Despite a demonstrated dependence on replicative polymerases, we show here that the replication of naked plasmid DNA in vitro does not require a functional ARS, the ORC, or the CDC6 gene product. As a result, our replication assay permits us to examine the effects of the Cdk1 and Cdc7 kinases on enzymatic events that take place downstream of the establishment and activation of the ORC-dependent initiation complex. By several approaches, we show that semiconservative DNA replication in vitro requires the activity of the Clb-Cdk1 kinase but not of the Dbf4-Cdc7 kinase. This finding suggests that B-type cyclin-dependent kinases play at least two roles in replication: in addition to regulating pre-RC assembly and disassembly, Clb-Cdk1 may regulate the replication machinery itself.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

Yeast strains, synchronization, isolation, and extraction of yeast nuclei. The genotypes of all strains used are listed in Table 1. GA-59, or congenic GA-811, was used as our standard wild-type strain, except as indicated otherwise. We have prepared extracts from a large variety of yeast strains and have detected no reproducible background-based variation in replication efficiency as long as the strains are wild type for cell cycle and replication factors. Several of the strains used here are nonetheless from the same background, notably, GA-112 and GA-1087, which are derived from the parent of GA-462. GA-161 and GA-315 are from the background of the original cdc collection of Hartwell et al. (31). All temperature-sensitive strains were checked for lethality at the restrictive temperature before and after culturing. The pep4 yeast strains were regularly tested by a plate-stain assay for carboxypeptidase Y activity (35).

In vitro replication reactions. Replication was carried out with 300 ng of either pGEM3Z() (hereafter called pGEM), pH4ARS (384-bp H4ARS Sau3AI fragment cloned into the SmaI site of the polylinker), or pRS424 (61) prepared according to a standard alkaline lysis method and purified over Qiagen columns or by banding on a CsCl gradient, avoiding exposure to UV light (59). At least 90% of each preparation was in the supercoiled form. Replication reactions were performed as described previously (6). For experiments performed at restrictive temperatures, the reaction mixture was incubated for 15 or 20 min at the elevated temperature prior to the addition of the template DNA. For control reactions, preincubation was done at 23 or 25°C. When needed, 500 µg of aphidicolin per ml, dissolved in dimethyl sulfoxide, was added, whereas control reactions received dimethyl sulfoxide alone. The replication reaction was stopped after 90 min (180 min for all 5-bromo-dUTP (BrdUTP) substitution experiments) at 25°C (or as indicated) with final concentrations of 0.3 M Na acetate (pH 5.2), 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, and 100 µg of proteinase K per ml. After incubation at 37°C for 30 min and the addition of a tRNA carrier, the DNA was extracted with phenol, precipitated, washed with 1 ml of 70% ethanol, dried, and resuspended in 20 µl of distilled water. For buoyant density analysis, the DNA was passed over a Sephadex G-50 spin column to eliminate free nucleotides. In all assays, the recovery of input DNA was quantified after the spin column step and before the CsCl gradient step by fluorescence readings and by separation of 25% of the reaction sample on an agarose gel (see Fig. 8C). Recovery of DNA in a given set of experiments usually varied less than 10%, but a correction factor for DNA recovery was introduced when semiconservative DNA synthesis was calculated in picograms of DNA synthesized or as fractions of the input template. Other modifications (substitution of BrdUTP for dTTP and buoyant density gradient analysis) are described elsewhere (6).

Analysis of semiconservative DNA replication. Two-dimensional (2D) gel electrophoresis (7), CsCl density gradient analysis, and the quantitation of these results with a Molecular Dynamics PhosphorImager or direct Cerenkov counting of incorporated radioactivity are described elsewhere (6). We have validated these methods by quantifying the recovery of DNA in the heavy-light (H-L) peak of the CsCl gradient by Southern blot analysis of slot blot gradient fractions or gradient fractions after agarose gel electrophoresis. Southern blot analysis is less quantitative than direct counting of incorporated ³²P-labeled deoxynucleoside triphosphates, because although a large fraction of the labeled DNA is in the H-L peak, a very small fraction of the total DNA shifts to this point in the gradient. Quantification by autoradiography and/or PhosphorImager techniques of the DNA recovered in the H-L peak from a range of experiments confirmed that between 0.1 and 2% of the input DNA was semiconservatively replicated (data not shown). The amount of label incorporated into the light-light (L-L) peak varies with the integrity of the supercoiled template and the endonuclease/ligase ratio in the extract and shows no positive correlation with the efficiency of semiconservative DNA replication. Indeed, the addition of nicked or linear template produces very high levels of incorporation into the L-L DNA peak and competes efficiently for semiconservative DNA replication (discussed in reference 6).

Immunodetection. Western blotting was performed with peroxidase-coupled secondary antibodies and enhanced chemiluminescence (Amersham). Rabbit antisera were used for the detection of Orc2p (gift of J. Diffley, ICRF, London, United Kingdom), p40^SIC1 (gift of R. Deshaies, Pasadena, Calif.), and topoisomerase II. Monoclonal antibodies were used to detect pol  and p86 (gifts of P. Plevani and M. Foiani, University of Milan, Milan, Italy), the Myc epitope (9E10), and tubulin TAT1 (gift of K. Gull, University of Manchester, Manchester, United Kingdom).

Kinase inhibition and activity assays. Kinase activity was assessed through measurement of histone H1 phosphorylation by established protocols (46). For the analysis of H1 kinase in nuclear extracts, the extracts were diluted to 100 ng/µl in 25 mM morpholinepropanesulfonic acid (MOPS) (pH 7.5)-15 mM MgCl₂-15 mM EGTA-1 mM DTT-60 mM -glycerophosphate-0.1 mM Na orthovanadate-15 mM p-nitrophenyl phosphate-1% Triton X-100 (kinase buffer). Then, histone H1 at 500 ng/µl and [-³²P]ATP at 0.1 µCi/µl were added to a total reaction volume of 20 µl. For purified-kinase assays, extracts were omitted. After 20 min at 23°C, the reaction was stopped by the addition of SDS sample buffer. Samples were run on a 10% acrylamide-SDS gel, which was then stained with Coomassie blue to quantify equal recovery of histone H1. Incorporated radioactivity was quantified with a PhosphorImager, and values were normalized to histone H1 recovery. It should be noted that the higher eukaryotic histone H1 is a relatively poor, nonphysiological substrate for the yeast Clb-Cdc28 kinase. In S-phase yeast extracts of a cdc28-4 strain isolated at the permissive temperature, there is little modification of histone H1 at 23°C, although these extracts are fully competent for modifying endogenous targets and stimulating replication in nuclei at permissive, but not restrictive, temperatures (54a). Thus, H1 kinase assays of yeast extracts give a relative but not an absolute measure of Cdc28 kinase activity.

Expression and purification of glutathione S-transferase (GST)-p40^SIC1-Myc-His₆ and CycB-Cdc2 kinase. Escherichia coli BL21 transformed with pLysS and the expression vector pGEX-4T-Sic1-Myc-His₆ (where His₆ is six histidines; containing the full-length SIC1 gene; gift of R. Feldman, Pasadena, Calif.) was grown at 30°C to an optical density at 600 nm of 0.5 in Luria broth with ampicillin (50 µg/ml) and chloramphenicol (20 µg/ml). The Sic1p fusion was induced with 0.8 mM isopropyl--D-thiogalactopyranoside (IPTG) for 90 min. Harvested cells were resuspended in 25 ml of 25 mM Tris-Cl (pH 7.6)-0.5 M NaCl-5 mM -mercaptoethanol-protease inhibitors (lysis buffer), and cells were quickly frozen in liquid nitrogen. The cells were thawed, lysed by sonication, and centrifuged for 20 min at 18,000 rpm in an SS34 rotor at 4°C. All subsequent steps were done at 4°C. Ni-NTA-agarose (2 ml; Qiagen) was added to the supernatant and mixed for 30 min. The lysate-resin mixture was poured into a column and washed with 5 volumes of lysis buffer plus 10% glycerol and 20 mM imidazole and then with 2 volumes of 25 mM Tris-Cl (pH 7.6)-0.2 M NaCl. Two elutions were carried out with 5 ml of the latter buffer plus 0.5 M imidazole. The eluates were pooled and adjusted to 1 mM EDTA and 1 mM DTT. All subsequent buffers contained these two reagents. Glutathione-agarose beads (1 ml; Sigma) were added and mixed with the eluate for 30 min. The beads were washed four times with 25 mM Tris-Cl (pH 7.6)-0.5 M NaCl and twice with 50 mM Tris-Cl (pH 7.6)-0.15 M NaCl, and elution was carried out with 3 ml of 50 mM Tris-Cl (pH 7.6)-0.15 M NaCl-10 mM reduced glutathione. Dialysis was carried out for 12 h against 25 mM Tris-Cl, (pH 7.6)-0.15 M NaCl-10% glycerol (dialysis buffer). Aliquots were frozen in liquid nitrogen and stored at 80°C.

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Nuclear extracts from yeast cells synchronized in S phase promote aphidicolin-sensitive, semiconservative replication of primer-free, supercoiled plasmids in vitro, as monitored by one-dimensional and 2D gel electrophoresis and by buoyant density analysis of the newly synthesized DNA following incorporation of the derivatized nucleotide BrdUTP (6). The reaction is dependent on DNA pol  and the pol -primase complex, and we have been able to detect putative Okazaki fragments under ATP-limiting conditions (6). In contrast to supercoiled templates, nicked or linear plasmid DNA incorporates BrdUTP through a repair mechanism that does not cause a shift in density to that of fully substituted H-L DNA, as monitored on CsCl gradients. The replication reaction is independent of the recombination-promoting factor Rad52p but requires the activity of the Dna2p helicase (6). Here we examine the dependence of our replication system on the ARS consensus, the pre-RC, and kinases that are known to promote S phase in vivo.

The replication of plasmid DNA in yeast nuclear extracts is ARS independent. Highly supercoiled preparations of bacterially produced plasmids (pGEM and pH4ARS; i.e. the same vector carrying the 384-bp H4ARS fragment) were incubated in either the presence or the absence of aphidicolin in an S-phase nuclear extract isolated from a protease-deficient yeast strain (GA-59; see Materials and Methods). After 90 min of incubation in the presence of [-³²P]dATP and other, unlabeled nucleotides, DNA was purified and separated on a nondenaturing agarose gel. As shown in Fig. 1A, lanes 1 and 3, the replication products of both pGEM and pH4ARS migrated as diffuse smears toward the origin of the gel. Except for a low level of repair incorporation, mainly into open circular DNA, the reaction was inhibited by aphidicolin and was fully dependent on the added template (Fig. 1A, lanes 2, 4, and 5).

View larger version (76K): [in this window] [in a new window]   FIG. 1.   DNA replication in nuclear extracts is ARS independent. (A) pH4ARS or pGEM (300 ng) was incubated in a GA-59 S-phase nuclear extract with all nucleotides and [-32P]dATP for 90 min at 25°C as described in Materials and Methods. Shown is an autoradiograph of the replicated DNA separated on a 0.8% agarose gel. Lanes 1 and 3, complete reactions with pH4ARS and pGEM, respectively. Lanes 2 and 4, like lanes 1 and 3 but with the addition of aphidicolin (Aph) at 500 µg/ml. Lane 5, no DNA added OC, open circular; SC, supercoiled. (B and C) Replication reactions were carried out as described for lanes 1 and 3 in panel A. The DNA was extracted, digested with EcoRI, and visualized by neutral-neutral 2D gel electrophoresis (7). Autoradiograms of dried gels are shown; the Y arc and one linear molecule (ln) are indicated. (D) For BrdUTP substitution, replication reactions were performed for 3 h at 23°C with 300 ng of supercoiled pH4ARS or pGEM in a GA-59 S-phase extract. BrdUTP replaces dTTP, and [-32P]dATP is added as a tracer (see Materials and Methods). Replicated DNA was extracted and filtered with Sephadex G-50, and recovery was checked by analyzing 25% of the reaction on an ethidium bromide-stained gel. Following CsCl gradient centrifugation of the remaining DNA, gradient fractions were analyzed for refractive index () and 32P incorporation. The H-L peak corresponds to an  of 1.4040, consistent with the migration of fully substituted DNA. The L-L peak (, 1.4000) corresponds to unsubstituted molecules. (E) Replication reactions were carried out for 3 h with GA-59 S-phase nuclear extract and fully methylated pH4ARS (lane 2) or pGEM (lane 4) as described in Materials and Methods. Following the reactions, replicated DNA was digested with ScaI and DpnI and analyzed on a 1% agarose gel. Complete digestion was monitored by ethidium bromide staining of the gel (right panel) and comparison with the migration of methylated plasmid DNA (i.e., pH4ARS in lane 1 and pGEM in lane 3) digested with DpnI and end labeled by standard procedures (59). The pattern of radioactive bands scanned with a PhosphorImager from the dried gel is shown in the left panel. Numbers at left indicate base pair markers.

Initiation is not strictly localized to an ARS element. The experiments shown in Fig. 1 indicated that the replication of supercoiled templates does not require an ARS element and suggested that sites of initiation might be dispersed along the plasmid sequence. This idea is consistent with the fact that we did not observe the arc indicative of a localized bubble and only rarely detected the very top of the bubble arc on 2D gels of linearized products (see Fig. 3B for an example). Since our template was small and the replication fork progresses quickly (3.7 kb/min in vivo) (25), we surmised that by assaying after 90 min, we might be favoring the detection of late or stalled RIs. However, pooling of aliquots taken at 2.5-min intervals between 0 and 15, 17.5 and 30, and 32.5 and 45 min failed to increase the bubble arc intensity (data not shown). The absence of the bubble arc was not due to inappropriate linearization of the plasmid, since digestion with EcoRI (which is adjacent to the ARS consensus of pH4ARS) or with ScaI (which cuts at a point 180° from the ARS consensus) yielded the same RIs on 2D gel analysis (Fig. 2A). Despite this result, a pronounced arc corresponding to a bubble within a circle was detected when nonlinearized replication products were analyzed on 2D gels (see reference 6). We can reconcile these results with two models: (i) initiation in this system is a poorly localized event or (ii) initiation is restricted to a specific site, but during digestion and reisolation of the replicated plasmid, bubble structures are artifactually converted to asymmetric Y arcs by breakage near the fork.

View larger version (55K): [in this window] [in a new window]   FIG. 2.   Initiation does not map specifically to the ARS consensus. (A) The replication reaction mixture described in Fig. 1A was scaled up, so that 600 ng of pH4ARS and 120 µg of wild-type S-phase extract (GA-59) were used. The recovered DNA was linearized with EcoRI or ScaI, and products were analyzed by 2D gel electrophoresis. Autoradiographs of dried gels are shown. Maps of the plasmids indicating the sites of relevant restriction enzymes are shown. (B) The helical stability of the pH4ARS plasmid (G values) was calculated as described by Marilley and Pasero (43). A linear map of the plasmid is shown under the graph; DNA-unwinding elements (66) are indicated by grey boxes and H4ARS is indicated by an empty box. D, DraI; H, HindIII. These sites were used to quantify label incorporation into appropriately restricted DNA (see the text).

Replication of plasmid DNA in S-phase extracts does not require components of the pre-RC. It has been proposed that the S-phase Cdk1 kinases modify components of the pre-RC, preventing their reassembly into a complex competent for reinitiation (12; reviewed in reference 47). Thus, the lack of origin dependence for initiation could reflect an inhibition of pre-RC assembly, the absence of ORC, or even the promiscuous binding of ORC to multiple sites in the plasmid. To ascertain whether ORC is present in our extracts, we probed for the presence of the 72-kDa subunit, Orc2p, by Western blotting. Although a large fraction of this protein was lost in an insoluble fraction that was removed by centrifugation after dialysis of the extract, Orc2p was readily detected in the nuclear extract used for replication (Fig. 3A, compare Pel and Ex). Moreover, Orc2p and other replication enzymes, such as pol , p86, and topoisomerase II, were stable after incubation at 37°C, mimicking the replication assay conditions for temperature-sensitive extracts (Fig. 3A).

View larger version (94K): [in this window] [in a new window]   FIG. 3.   The orc2-1 mutation does not abrogate the appearance of RIs during plasmid replication in vitro. (A) Immunoblotting was performed by standard procedures (59). In the first panel, Orc2p in 25 µl of GA-59 S-phase nuclear extract (Ex) and in the insoluble material (Pel) recovered from the same volume of extract (i.e., a fraction rendered insoluble by dialysis) was detected with an anti-Orc2p antibody. The insoluble fraction has the appearance of nuclei under the microscope and may reflect residual structures not removed by centrifugation in high-salt conditions. In the second panel, nuclei from orc2-1 cells (GA-462) grown at 23°C were incubated at either 25 or 37°C for 30 min, and 25 µg of each preparation was probed for Orc2p. In all other panels, 60 µg of GA-59 S-phase extract was incubated for 90 min at either 25 or 37°C as indicated and subsequently analyzed by Western blotting with monoclonal antibody 24D9 against DNA pol  (56), monoclonal antibody 6D2 against p86 (28), or a rabbit polyclonal antibody against topoisomerase II. Secondary antibodies were peroxidase coupled and detected by chemiluminescence. Molecular weight standards (in thousands) are indicated at the left. (B) pH4ARS (300 ng) was incubated at 25 or 37°C in an S-phase nuclear extract prepared from orc2-1 cells (GA-462) for 90 min in the presence of [-32P]dATP as described in the legend to Fig. 1A. Reaction products were separated by neutral-neutral 2D gel electrophoresis (see Materials and Methods). An arrowhead indicates the top of the putative bubble arc, exposure times are indicated in the lower left corners, and the 2D gel pattern is explained on the right. oc, open circular; n, mass of one plasmid.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Semiconservative DNA replication in nuclear extracts requires neither Cdc6p nor intact ORC. (A) BrdUTP-containing replication reactions (Fig. 1D) were performed in parallel with 300 ng of either pH4ARS (open circles) or pGEM (filled circles). Incubation was done at 23°C for 3 h with a GA-462 (orc2-1) S-phase extract prepared at the permissive temperature. The reactions were preceded by 15 min of preincubation at 23°C of all components except DNA. Following replication, the DNA was purified, recovery was quantified, and 75% of the reaction was separated on a CsCl density gradient. All fractions were monitored for refractive index and 32P incorporation. Peaks corresponding to the migration of H-L and L-L molecules are indicated. (B) Reactions identical to those in panel A were performed, except that reaction mixtures were preincubated at 35°C and then cooled to 23°C before the addition of DNA. (C and D) Similar BrdUTP-containing replication reactions were performed with 300 ng of supercoiled pH4ARS in GA-59, GA-462 (orc2-1) or GA-315 (cdc6-1) S-phase nuclear extracts, all prepared at the permissive temperature. Incubation was done for 3 h at either 23°C (C) or 35°C (D). Replicated DNA was separated on CsCl density gradients. The expected migration of H-L and L-L molecules is indicated by arrowheads. Incorporation was lower at 35°C for all strains tested (see the y axis for counts per minute).

Semiconservative DNA replication is impaired by a conditional mutation in CDC28 but not in CDC7. In our initial studies of replication in vitro, we readily observed Y arcs on 2D gels following reactions with S-phase nuclear extracts but not with nuclear extracts prepared from pheromone-arrested G₁-phase cultures (6) (see also Fig. 6A and B). Western blotting of equivalent aliquots of nuclear extracts from either unsynchronized cells or cells synchronized in G₁, S, or M phase indicated that the levels of many replication-related proteins did not vary significantly through the cell cycle (data not shown; pol , p180, and p86 subunits, PCNA, RPA, topoisomerase II, and the third subunit of replication factor [RF-C] were tested). In view of this result, the obvious candidates for rendering our in vitro replication reaction cell cycle dependent were the S-phase-promoting kinases Cdc28p and Cdc7p (34, 48).

View larger version (29K): [in this window] [in a new window]   FIG. 5.   Replication in vitro is sensitive to the loss of Cdc28 but not Cdc7 kinase. (A) BrdUTP-containing replication reactions were carried out for 3 h at 23, 30, or 35°C (for cdc7-1 only) as described in Materials and Methods with 40 µg of S-phase nuclear extract from either GA-112 (cdc28-4) prepared at the permissive temperature or GA-85 (cdc7-1) synchronized by a temperature shift (see Materials and Methods). All reactions were performed in parallel with 300 ng of pH4ARS as a template, and equal recovery of the plasmid was checked prior to CsCl gradient analysis. Cerenkov counts of the fractions from the CsCl gradients are shown. Fractions with refractive indices appropriate for H-L and L-L DNAs are indicated. The increase in the replication efficiency of the cdc7-1 extract at 30°C compared to that at 23°C was also observed for the wild-type extract (data not shown). (B) Parallel reactions like those in panel A were performed at 23 and 30°C for 3 h with 40 µg of a 1:1 mixture of cdc28-4 and cdc7-1 extracts. DNA recovery was monitored. After CsCl gradient analysis, the relative levels of replication supported by each extract at 23 or 30°C or by the 1:1 mixture were quantified by integration of the H-L replication peak. Relative values are expressed as arbitrary units. (C) Histone H1 kinase assays were performed at the indicated temperatures as described in Materials and Methods with equal amounts of the cdc28-4 and cdc7-1 extracts (see panel A). Histone H1 is indicated. Although we did not detect H1 modification in the cdc28-4 extract at 23°C, this extract showed temperature-sensitive promotion of replication in G1-phase nuclei in vitro (see Materials and Methods).

Cell-cycle-dependent stimulation of replication correlates with alterations in the p40^SIC1 inhibitor. The protein p40^SIC1 is a physiological inhibitor of the Clb5-Cdc28 complex that accumulates during G₁ phase and that becomes phosphorylated, ubiquitinated, and degraded at the G₁/S transition point (14, 26, 36). If the critical, limiting activator for semiconservative replication in vitro is the Clb5-Cdc28 kinase, we reasoned that extracts prepared from cells that accumulate p40^SIC1 should be inactive for replication. If the required Clb-Cdk1 activity is not restricted to Clb5-Cdc28, then M-phase nuclear extracts may actively support replication. To test these hypotheses, we prepared a late-G₁-phase nuclear extract from cdc4-1 cells (GA-1087) arrested at 37°C due to the inactivation of the complex that targets p40^SIC1 for degradation (14, 26). This extract was compared to a wild-type S-phase extract and a cdc16-1 M-phase extract prepared from cells blocked in mitosis by a similar temperature shift. The arrest by the temperature shift inactivates Cdc16p, which is needed to target B-type cyclins for degradation at the metaphase-anaphase transition (2). Replication reactions were performed in parallel at the permissive temperature, and H-L DNA was monitored by buoyant density analysis.

View larger version (39K): [in this window] [in a new window]   FIG. 6.   Replication efficiency and modification of P40SIC1 in nuclear extracts vary in a cell-cycle-dependent manner. (A) Quantitation is shown for integrations of the H-L peaks (black bars) from multiple BrdUTP substitution reactions performed for 3 h at 23°C with 40 µg of a G1-phase (cdc4-1; GA-1087), an S-phase (GA-59), or an M-phase (cdc16-1; GA-161) nuclear extract and 300 ng of template as described in Materials and Methods. Buoyant density analysis was performed on replicated DNA after plasmid recovery was checked. For the cdc4-1 G1-phase extract and the GA-59 S-phase extract, five reactions were performed under identical conditions, and the area of the H-L replication peak following CsCl gradient analysis of each was quantified in arbitrary units (for the cdc4-1 extract, the mean was 13.2 [standard deviation, 7.6]; for the GA-59 extract, the mean was 50 [standard deviation, 8.7]). Three reactions with the cdc16-1 M-phase extract were performed in parallel (mean, 54.6 [standard deviation, 14.4]). Comparative replication reactions were always performed with identical template preparations. The relative H1 kinase activities for equal aliquots of the extracts are indicated by white bars. (B) Replication reactions were performed as in panel A with 40 µg of a G1-phase nuclear extract (GA-1087), 40 µg of a wild-type S-phase extract (GA-360), or a mixture of 20 µg of each. Reactions were performed in parallel for 3 h at 23°C with 300 ng of pGEM. The H-L peaks of the CsCl gradients were integrated (black bars). Histone H1 kinase assays were done with the cdc4-1 G1-phase extract (GA-1087), the S-phase nuclear extract (GA-360), and a 1:1 mixture of the two. An autoradiograph of the kinase assays is shown in the inset, quantiation in arbitrary units is shown by white bars. (C) Immunodetection of endogenous p40SIC1 in a series of replication reactions allowed to proceed at 23°C for the times indicated. Reaction mixtures contained 40 µg of a wild-type S-phase extract (GA-59), 40 µg of the cdc4-1 G1-phase extract (GA-1087), or a mixture of 20 µg of each and 300 ng of pGEM but no radioactive dATP. Loading buffer (4×; 200 mM Tris-Cl [pH 6.8], 400 mM DTT, 8% SDS, 0.4% bromophenol blue, 40% glycerol) was added to equal samples taken at the indicated time. p40SIC1 was detected by immunoblotting with a polyclonal anti-p40SIC1 serum. The lower band of P40SIC1 was presumably already modified by Cln/Cdc28, and a rapid and complete shift to a slower mobility was detected during incubation in the S-phase extract or in the mixture of extracts but not in the G1-phase extract. The asterisk indicates an unrelated cross-reacting polypeptide that served as an internal loading standard. As expected, levels of P40SIC1 were lower in the S-phase extract. (D) Bacterially expressed GST p40SIC1-Myc-His6 (GST-p40SIC1) (see Materials and Methods) was added at a concentration of 0.8 ng/µl to replication reactions carried out as described for panel C but with cdc4-1 (GA-1087) G1-phase or cdc7-1 (GA-85) or cdc28-4 (GA-112) S-phase nuclear extracts prepared as described in Materials and Methods. DNA was omitted from one of the reactions with the GA-85 S-phase extract (DNA). Immunoblotting was carried out as described for panel C but with an anti-Myc monoclonal antibody (9E10). The 0 time point represents input GST-p40SIC1-Myc-His6. (E) Integration of the H-L replication peaks determined from quantitative buoyant density analysis of BrdUTP-containing replication reactions is shown in arbitrary units. These reactions were performed for 3 h at 23°C with 300 ng of pRS424 DNA and 40 µg of GA-112 (cdc28-4) S-phase or GA-1087 (cdc4-1) G1-phase nuclear extract, either with (grey bars) or without (white bars) the addition of 2 U of baculovirus-expressed Clb5-Cdc28 (see Materials and Methods). To the right, Western blot analysis for p40SIC1 was performed as described for panel C but with 40-µg aliquots of the GA-112 (cdc28-4) S-phase or GA-1087 (cdc4-1) G1-phase nuclear extract. The analysis showed that the latter had higher levels of p40SIC1. As shown in panel C, p40SIC1 in the G1-phase extracts had faster mobility.

Cdk1p is necessary and sufficient to stimulate semiconservative DNA replication in G₁-phase extracts. To confirm that the minimal replication level observed in the cdc28-4 S-phase extract reflected the absence of Clb5-Cdc28 kinase activity, we complemented this extract with highly purified baculovirus-expressed Clb5-Cdc28 and performed parallel replication reactions at 23°C (see Materials and Methods). The addition of 2 U of Clb5-Cdc28 was sufficient to double the efficiency of semiconservative plasmid replication in the cdc28-4 mutant extract (Fig. 6E). Lower levels of enzyme resulted in less stimulation, while the addition of 30 or 100 U no longer increased the recovery of H-L DNA (data not shown) (see below). In contrast, 2 U of recombinant Clb-Cdc28 kinase was unable to stimulate replication in the cdc4-1 extract (Fig. 6E). This result was presumably due to the high level of p40^SIC1 in the cdc4-1 extract (see Western blot of equivalent extract aliquots in Fig. 6E), which probably inhibited the added kinase (see below).

View larger version (34K): [in this window] [in a new window]   FIG. 7.   Addition of mitotic CycB-Cdc2 stimulates replication in both S-phase and G1-phase extracts. (A) Histone H1 kinase assays were performed at 23°C as described in Materials and Methods with 20 U of purified recombinant Clb5-Cdc28 or CycB-Cdc2 and with increasing concentrations (0.16, 0.8, and 4 µM) of either bacterially expressed GST-p40SIC1-Myc-His6 (GST-p40SIC1) or GST alone in a 10-µl reaction volume. Histone H1 is indicated. (B and C) CsCl gradient analyses of BrdUTP-containing replication reactions performed in parallel with 300 ng of pRS424 and 40 µg of either wild-type (wt) S-phase (GA-811) or cdc4-1 G1-phase (GA-1087) nuclear extract for 3 h at 23°C. Either 10 U of baculovirus-expressed Xenopus CycB-Cdc2 or an equivalent volume of the corresponding fraction (control fr.) from mock-infection Sf9 cells was added to the replication reactions as indicated. The recovery of DNA was checked prior to CsCl gradient analysis (see lanes 5 to 9 of Fig. 8C). Integration of the H-L peaks is shown to the right of the gradient profiles and is given in absolute picograms of replicated DNA. (D) Xenopus CycB-Cdc2 does not stimulate the degradation of P40SIC1 during replication reactions. Replication reactions were performed as described for panel B with 40 µg of a cdc4-1 (GA-1087) G1-phase nuclear extract, either with (+) or without () the addition of 10 U of baculovirus-expressed Xenopus CycB-Cdc2. Radioactive nucleotide was omitted. Where indicated (+S), the reaction mixtures contained 20 µg each of the G1-phase (GA-1087) and wild-type S-phase (GA-811) extracts. Samples taken after 1 or 2 h and input G1-phase extract (0 time point) were analyzed by Western blotting for endogenous p40SIC1 as described in the legend to Fig. 6. The asterisk indicates a cross-reacting polypeptide that was used as a loading control.

CycB-Cdc2 kinase stimulates replication in cdc28-4 and wild-type S-phase extracts. The observation that the cdc28-4 S-phase extract was deficient for semiconservative DNA replication suggested that Clb-Cdc28 kinase activity is required continuously during DNA synthesis and not simply to inactivate an inhibitor of replication at the G₁/S boundary. Since our S-phase extracts still contained a low level of p40^SIC1 (Fig. 6), it seemed possible that Clb-Cdk1 activity was limiting for replication even in the wild-type S-phase extract. To test this possibility, we added 10 U of purified Xenopus CycB-Cdc2 to the standard replication assay with a wild-type S-phase extract. BrdUTP incorporation was monitored by buoyant density analysis, and the recovery of DNA was systematically quantified (see Fig. 8C). The addition of 10 U of CycB-Cdc2 to the wild-type S-phase extract stimulated replication over the control level (2.5-fold) (Fig. 7C), while the addition of a control fraction had no effect (data not shown). As an internal standard, replication in the cdc4-1 extract was assayed in parallel, and the H-L peaks were quantified by integration (Fig. 7B and C). In conclusion, Xenopus CycB-Cdc2 stimulated replication in both S-phase and cdc4-1 G₁-phase nuclear extracts.

View larger version (26K): [in this window] [in a new window]   FIG. 8.   Xenopus CycB-Cdc2 activates plasmid replication in a cdc28-4 S-phase nuclear extract. (A) Buoyant density analysis was done for BrdUTP-containing replication reactions performed in parallel with those shown in Fig. 7C. Reactions were carried out with cdc28-4 (GA-112) S-phase nuclear extracts and 300 ng of pRS424 for 3 h at 23°C. Reaction mixtures contained either 10 U or recombinant Xenopus CycB-Cdc2 kinase (grey squares) or an equivalent amount of kinase buffer (black circles). DNA recovery was checked prior to CsCl gradient analysis and is shown in lanes 1 and 3 of panel C. The expected migration of H-L and L-L molecules is indicated by arrowheads. (B) BrdUTP-containing replication reactions were identical to those in panel A, except that the Xenopus CycB-Cdc2 kinase was pretreated with either GST-Cip1 beads (black diamonds) (see Materials and Methods) or GST beads (white triangles). Similar results were obtained when the kinase was inactivated by reductive alkylation and added directly to the reaction mixtures (see Materials and Methods). (C) Systematic analysis performed after each replication reaction; 25% of the recovered DNA was analyzed and quantified on an 0.8% agarose gel prior to loading of the remaining 75% on CsCl gradients. The ethidium bromide-stained plasmid was detected by UV fluorescence and quantified by scanning. The samples shown on the gel include, among others, the products from reactions analyzed in Fig. 7 and panels A and B. (D) Kinase activity as monitored by the phosphorylation of histone H1 by untreated recombinant Xenopus CycB-Cdc 2 kinase or kinase treated with either GST-Cip1 bound to glutathione-agarose beads or GST bound to glutathione-agarose beads. The relative kinase activity in equal portions of cdc16-1 (GA-161) M-phase and cdc4-1 (GA-1087) G1-phase extracts is also shown as a reference.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

Coordination between the cell cycle and the initiation of DNA replication in budding yeast depends primarily on the assembly of proteins at the origin of replication and trans-activating cyclin-dependent kinases. The pre-RC that assembles in G₁ phase is altered as origins fire and usually requires passage through mitosis to render it competent for reinitiating DNA replication (47, 48). The exact composition of the proteins that form the pre-RC is still unclear, and much remains to be learned about how S-phase-promoting kinases stimulate both the initiation and the subsequent elongation steps of DNA replication. The possibility that pre-RC-independent initiation can occur in yeast has not been excluded, since orc mutants do progress partially through the S phase at the restrictive temperature (3), and a chromosome III fragment lacking functional ARS elements can be replicated (48a). Here we examined an in vitro replication system from budding yeast cells that initiates DNA replication in the absence of pre-RC formation. We demonstrated a cell cycle dependence of this reaction, which is most simply explained as a requirement for CycB-Cdk1 activity. Our results suggest that the S-phase Clb-Cdc28 complex does more than dismantle the ORC-dependent pre-RC at the G₁/S transition to promote genomic DNA replication.

The semiconservative replication of supercoiled plasmid templates in vitro requires the activity of the pol -primase complex, pol , and the Dna2 helicase (6). Consistently, the reaction requires ribonucleotides and can be inhibited both by antibodies that inactivate pol  and by aphidicolin, suggesting that this system retains mechanistic aspects of genomic DNA replication. However, unlike replication in intact cells or the replication of nuclear templates in vitro (53), initiation occurs in the absence of an ARS consensus and independently of two essential components of the pre-RC, ORC and Cdc6p (Fig. 3 and 4) (64). Preliminary depletion data suggested that the reaction is MCM independent as well (31a). These unconventional initiation events may reflect the ability of pol -primase to associate with chromatin in a pre-RC-independent manner, as reported by Desdouets et al. (13), yet these authors found the chromatin association to be inhibited by Clb-Cdk1 kinases (see below). Alternatively, gene conversion-type events which lead to chromosome replication may occur in vitro, although the appearance of RIs in our system does not require RAD52 and is not stimulated by the addition of homologous single-stranded DNA (6).

In S-phase extracts from an orc2-1 mutant, we still detected semiconservative plasmid replication at the nonpermissive temperature, although the efficiency was reduced. Since this finding was true even for plasmids that lacked an ARS consensus, we assumed that it reflected either the temperature-induced lability of another component or indirect interference in initiation due to dissociation of the ORC. Consistent with this latter hypothesis, we also detected the inhibition of plasmid replication in S-phase extracts when baculovirus-expressed ORC was titrated into the reaction (31a). Since this finding also occurred with templates lacking an ARS consensus, it suggests that an excess of "free" ORC can repress replication. Surprisingly, in preliminary experiments, we found that the repressive effect of exogenous ORC was reproducibly counteracted by preincubating the complex and template with a G₁-phase extract prior to adding the S-phase extract to promote replication. Such results may be the first steps in reconstituting a functional pre-RC, which may require the assembly of ORC, Cdc6p, Dbf4-Cdc7p, MCM, and perhaps nucleosomes on templates in extracts that lack Clb-Cdc28 activity (15, 48). A reconstituted origin may then be able to fire in an origin-specific manner, like the initiation observed in isolated G₁-phase nuclei (53).

Because the replication that we detected in extracts occurred in the absence of pre-RC formation, we were surprised to find it subject to cell cycle controls. While this finding might reflect the availability of a limiting replication enzyme, we detected no major variations in the levels of the common replication factors throughout the cell cycle. With both conditional mutants and reconstitution assays, we showed here that CycB-Cdk1 activity but not that of Dbf4-Cdc7 kinase, appeared to be limiting for ORC-independent initiation events in vitro. Notably, the inefficient plasmid replication supported by the cdc28-deficient extract could be complemented by adding an equivalent amount (2 U) of purified Clb5-Cdc28 complex or 10 U of Xenopus CycB-Cdc2 complex. Titrations showed less stimulation when smaller amounts were added and inhibition when a vast excess of kinase was added (30 to 100 U) (data not shown). If this inhibition is not artifactual, it may mean that optimal kinase levels are carefully balanced in the cell and that excessively high Clb-Cdk1 levels play a role in downregulating DNA polymerase activity. Such fine-tuning of DNA replication may be one function of the CLB3 and CLB4 gene products that accumulate during S phase (48). Although the interpretation of these results requires further analysis, there are many precedents for differential phosphorylation patterns of a given protein regulating activity in opposite directions (see the discussion of DNA pol -primase complex below and reference 67).

Activation of a late-G₁-phase extract by Xenopus CycB-Cdk1. A G₁-phase extract from a cdc4-1 strain which accumulated high levels of Cln/Cdc28-modified p40^SIC1 at a restrictive temperature supported very low levels of semiconservative plasmid replication. In S-phase extracts, p40^SIC1 levels were much lower, and when G₁- and S-phase extracts were mixed, we saw a shift in inhibitor mobility consistent with a rapid phosphorylation of the already modified polypeptide. This further modification was probably due to Clb-Cdc28, for the shift of recombinant p40^SIC1-Myc-His₆ occurred in S-phase extracts from wild-type and cdc7-1 cells but not from cdc28-4 cells (Fig. 6D).