Correspondence to: Peter K. Jackson, Department of Pathology and Department of Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Dr., Palo Alto, CA 94305-5324. Tel:(650) 498-6872 Fax:(650) 725-6902 E-mail:pjackson{at}stanford.edu.
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Abstract |
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Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin ECdk2 to DNA. We find that cyclin E binds the NH2-terminal region of Cdc6 containing CyArg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin ECdk2 for chromatin binding, and fail to rescue replication in cyclin Edepleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin ECdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin ECdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin ECdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin BCdc2, but not the polo-like kinase 1, remove cyclin ECdk2 from chromatin. Rebinding of hyperphosphorylated cyclin ECdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinasedirected Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin ECdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.
Key Words: cyclin-dependent kinases, origin recognition complex, DNA replication, Cdc6, Cdc14
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Introduction |
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The requirements for determining the timing and origin selection for eukaryotic DNA replication are now being intensively investigated. In yeast, origin selection requires the origin recognition complex (ORC)1 to bind initiation sites on DNA (
The cyclin ECdk2 complex is essential for timing initiation of DNA replication (200-fold within the nucleus after nuclear assembly (
How cyclin ECdk2 promotes DNA replication remains unclear, because we do not know its relevant substrates, how those substrates are selected, or how phosphorylation by cyclin ECdk2 changes their ability to promote replication. Candidates for cyclin ECdk2 substrates have been described, including the protein NPAT (
The ability of cyclinCdk complexes to select their specific substrates is determined in part by binding of the cyclin to regions on the substrate. The crystal structure of human cyclin ACdk2 bound to the inhibitor/substrate p27Kip1 defined a region of the cyclin A protein that interacts directly with p27 (
We were interested in further understanding the mechanisms governing cyclin ECdk2 control of DNA replication. Because cyclin ECdk2 likely phosphorylates chromatin-associated prereplication proteins, we speculated that cyclin E might function on chromatin. Here, we show that cyclin ECdk2 associates with chromatin in three phases and that this association in the first phase depends primarily on the prior recruitment of the ORCCdc6 complex. We further show that the cyclin ECdk2Cdc6 interaction is a direct association mediated by the MRAIL motif in cyclin E and the RXL motif, and possibly another site in the NH2 terminus of Cdc6, and that this interaction is essential for the initiation of DNA replication. In the second phase, cyclin ECdk2 accumulates on chromatin as replication proceeds, potentially explaining the ability of cyclin ECdk2 to block rereplication. We find this accumulation requires polymerase activity. In the third phase, the cyclin Echromatin interaction is abolished in mitosis and reestablished upon the exit from mitosis, thereby allowing a new round of replication. We have found that cyclin BCdc2 and, to some extent, mitogen-activated protein (MAP) kinase are capable of phosphorylating cyclin E in mitosis and removing it from chromatin, and that Cdc14, a phosphatase essential for the exit from mitosis, is capable of reversing the mitotic phosphorylation of cyclin E and allowing it to rebind chromatin in G1. Thus, the cell cycleregulated three-phase association of cyclin E with its chromatin receptor may help explain the coordination of its functions in initiating replication, blocking rereplication, and relicensing origins.
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Materials and Methods |
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Preparation of Xenopus Egg Extracts and Sperm Nuclei
For interphase extracts, dejellied eggs were rinsed in ELB (250 mM sucrose, 2.5 mM MgCl2, 50 mM KCl, 10 mM Hepes, pH 7.7, 1 mM DTT, 50 µg/ml cycloheximide, 10 µg/ml cytochalasin D), and centrifuged (13,000 rpm, 10 min). Cytosol was recentrifuged (24,000 rpm, 10 min), and the supernatant was removed with a syringe and kept on ice; the second spin significantly improved replication efficiency. Cycling extracts were made similarly, except that eggs were activated by the calcium ionophore A23187 (Sigma-Aldrich), and cycloheximide was omitted from the buffer (
Sedimentation Assays to Isolate Assembled Chromatin from HSS and Low Speed Supernatants
HSS Reactions.
HSS for chromatin assembly was made as described above. Reactions were carried out by incubating 20 µl of interphase HSS with 20 ng of sperm DNA or 1 µg of DNA, diluted to 50 µl with XB2 (XB with 2 mM MgCl2). In some experiments, baculovirus-expressed cyclin ECdk2 was added to 200 nM. Inhibitors or recombinant proteins were preincubated with HSS for 15 min before DNA template addition. Upon DNA addition, reactions were incubated (30 min, 22°C), stopped by dilution (150 µl of cold XB2), layered on a 400-µl cushion (1.1 M sucrose in XB2), and spun (11,000 rpm, 30 min, 4°C) in a SW50.1. The gradient interface was washed with XB2 to remove unpelleted material, and sample buffer was added to the pellet for SDS-PAGE.
LSS Reactions.
Low speed supernatant (LSS) was supplemented with an energy regenerating system before sperm addition (1,000 sperm/µl). Samples were incubated (23°C) for the indicated times, diluted with five volumes of cold ELB, layered over a 0.5-M sucrose cushion, and centrifuged in a Beckman 152 microfuge (20 s). Pelleted nuclei were resuspended in sample buffer and analyzed by Western blotting. Chromatin was extracted from a duplicate set of assembled nuclei by adding 10 volumes of chromatin extraction buffer (50 mM KCl, 50 mM Hepes, pH 7.7, 5 mM MgCl2, 5 mM EGTA, 2 mM ß-mercaptoethanol, 0.5 mM spermidine, 0.15 mM spermine, 0.1% NP-40), mixing gently, and leaving on ice for 30 min, before respinning the tubes as above. Similar assays show the association of replication proteins with chromatin templates (
Samples treated with mitotic kinases were assembled in LSS (1 h) and murine MAP kinase, human cyclin BCdc2 (1 U each; New England Biolabs, Inc.), or glutathione S-transferase (GST)XPlk1 (a gift from Jan-Michael Peters, Institute of Molecular Pathology, Vienna, Austria) were added for 10 min. Chromatin fractions were isolated as above.
Replication Assays
10 µl of cycloheximide-stabilized interphase extract was mixed with 35 ng of sperm, and replication assays were performed and quantitated as described (
Phosphorylation/Dephosphorylation Reactions
2.5 µg of bacterially expressed, purified GSTXcyclin E was incubated with 1 U of MAP kinase, cyclin BCdc2, GSTPlk1, or baculovirus-expressed cyclin ECdk2 in kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 100 µM ATP) in the presence of 0.15 µCi of [32P]ATP. After 30 min at 30°C, half of each sample was removed and supplemented with 2 µM GSTCdc14. Cdc14-treated and -untreated samples were incubated (30 min, 30°C) before stopping reactions with sample buffer, resolving by SDS-PAGE, and visualizing phosphorylated GSTcyclin E by autoradiography.
Calculation of the Number of Cyclin E Molecules Per Origin
The concentration of cytosolic cyclin ECdk2 required for binding to chromatin was estimated by adding baculovirus-expressed cyclin ECdk2 to DNA in cyclin Edepleted HSS. The number of molecules per origin represented by this binding event was calculated by determining the percentage of cyclin E that bound to DNA by quantitative Western blotting. Assumptions include that the number of origins per nucleus equals 105 (/liter nuclei) = (9 x 107 molecules/nuclei). Because
0.1% of the cyclin E from HSS binds to chromatin, we estimate that
1 x 105 molecules/nuclei per 105 origins/nucleus =
1 molecule cyclin E/origin.
To determine the maximum capacity of chromatin for cyclin E, known amounts of baculovirus cyclin ECdk2 were titrated into cyclin Edepleted LSS extracts and chromatin-associated cyclin E, measured by quantitative Western blot. The maximal level was roughly equal to the amount of endogenous cyclin E bound to chromatin immediately before mitosis.
In Vitro Binding Assays
GST fusion proteins of either human p21N, human p21C, human p27, XCdc6N, XCdc6C, or human Cdc14, added to a concentration of 1 µM, were mixed with baculovirus-expressed Xcyclin EXCdk2 (0.4 µM) and diluted to 10 µl with XB-. Mixtures were incubated (1 h, 25°C), diluted with 90 µl of immunoprecipitation (IP) buffer (100 mM NaCl, 50 mM ß-glycerophosphate, 5 mM EDTA, 0.1% Triton X-100, pH 7.2), and spun (13,000 g, 10 min). Supernatants were added to glutathioneagarose and rocked (30 min, 4°C). Beads were washed with IP buffer, resuspended in sample buffer, and resolved by SDS-PAGE.
Mutants of Xcyclin E were created by PCR mutagenesis and verified by sequencing as M143A L147A W150A and L186A Q187A. RXL mutants of XCdc6 were engineered and verified as (a) R93A L94A L95A, (b) R165A L167A, and (c) R258A L260A.
In vitrotranslated (IVT) 35S-labeled cyclin E was expressed from pGEM3Zf+ using the TNT-coupled reticulocyte lysate systems (Promega).
Purification of the XORC Complex from Xenopus laevis Extract
ORC was purified 500-fold from HSS made from the eggs of 50 frogs (
Immunodepletion and ATP Depletion
Immunodepletions were performed by binding crude (Xcyclin E) or affinity-purified (XCdc6) rabbit sera to protein ASepharose beads for 1 h. Antibody beads were incubated with extract (2 x 45 min, 4°C) and then centrifuged (13,000 rpm, 10 min). Control depletions were performed with beads alone. ATP depletion was performed by adding hexokinase beads (Sigma-Aldrich), and residual ATP was determined to be <3% by luciferase assay.
Antibody Production and Purification
Purified GSTXORC2, GSTXORC1, GSTXcyclin E, and GSTXCdc6 were used to raise antisera in rabbits (Josman Immunoresearch). Affinity purification of antisera was performed by acid elution from MBP fusion proteins coupled to CNBr-activated Sepharose. Anti-Cdk2 antibodies have been previously described (
Production of Bacterially Expressed GST and MBP Proteins and Baculovirus-expressed Cyclin ECdk2 and Cdc6
GST and MBP fusion proteins were expressed in BL21 pLysS and purified over glutathione or amylose resins as described (
The following fragments were made as GST or MBP fusions: p21N, amino acids 190; p21C, amino acids 87164 (
Production of baculovirus-expressed His-XCdc6 was performed by infecting Sf9 cells with the XCdc6 virus (a gift from Bill Dunphy, California Institute of Technology, Pasadena, CA) and purifying over NiNTA resin (QIAGEN).
Baculovirus-expressed Xcyclin EHis-XCdk2 (a gift of Jim Maller, University of Colorado, Denver, CO) was produced by coinfection with His-XCdk2 virus (multiplicity of infection [MOI] = 10) and Xcyclin E virus (MOI = 15) to favor cyclin ECdk2 complex formation (1 mM).
Western Blotting
Western blotting was performed as described (
Online Supplemental Material
Deletion mutant analysis was used to map an NH2-terminal region of XCdc6 outside of the RXL motif that interacts with Xcyclin E. Supplemental Figure S1 depicts experiments assessing the ability of XCdc6 NH2-terminal deletion mutants to bind to cyclin E, become phosphorylated by cyclin ECdk2, and sustain replication. Figure S1 is available at http://www.jcb.org/cgi/content/full/152/6/1267/DC1
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Results |
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Cyclin ECdk2 Is Recruited to Chromatin after Nuclear Accumulation and Is Removed from Chromatin in Mitosis
To study the ordered events of DNA replication, we optimized an assay to isolate chromatin templates assembled within nuclei formed in LSS of Xenopus egg extracts. These cycling extracts recapitulate the events of the mitotic cell cycle in vitro. First, we separated sperm nuclei assembled in LSS from the cytosolic fraction by centrifugation (Fig 1 A). We extracted purified nuclei with chromatin extraction buffer and recentrifuged to separate nucleoplasmic proteins from tightly chromatin-associated proteins. Similar assays have been performed in several systems to study the association of replication proteins with chromatin templates (Materials and Methods). The amount of DNA replication completed at each time point is shown for reference (Fig 1 B). Because cyclin ECdk2 promotes DNA replication, we tested whether cyclin ECdk2 directly interacts with chromatin. We found that cyclin ECdk2 associated with chromatin assembled in cycling LSS extracts (Fig 1 A). In this first phase, cyclin ECdk2 was imported into the nucleus after nuclear assembly and bound to chromatin immediately after nuclear import, unlike ORC and Cdc6, which associated with chromatin before nuclear formation (Fig 1 A). Cyclin E became detergent-inextractible at the same time that MCMs appear in the detergent-extracted chromatin fractions (not shown).
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In a second phase, cyclin E continued to accumulate on chromatin throughout replication (Fig 1 A).
In a third phase, chromatin binding of cyclin ECdk2 was mitotically regulated. When cyclin BCdc2 kinase activity peaked (indicated by the triangle containing an M), cyclin ECdk2 was rapidly displaced from chromatin (Fig 1 A). Although we saw displacement of XORC1 and XORC2 later in mitosis (not shown), XORC2 appeared to be more stably associated with chromatin in early mitosis (Fig 1 A) when nuclear envelope breakdown was first initiated. Addition of the phosphatase 2A inhibitor okadaic acid to interphase extracts also induced the mitotic state (
A Chromatin Assembly Assay Shows That Cyclin E Associates with Chromatin with Kinetics Similar to ORC and Cdc6
To study the first phase of cyclin ECdk2 binding to interphase chromatin, we optimized an assay to isolate Xenopus sperm or DNA templates assembled in HSSs of interphase egg extracts (
DNA behave identically in all of our HSS assays, which were each repeated using both templates to verify results. The DNA templates used are noted in the figure legends. After chromatin assembly, reactions were overlaid on a sucrose cushion and chromatin isolated by sedimentation. The chromatin-associated proteins were resolved by SDS-PAGE and examined by Western blotting. The assay was optimized to ensure a high efficiency of isolating the chromatin templates (>95%) and to minimize nonspecific sedimentation of cytoskeletal proteins (Materials and Methods).
In this assay, ORC and Cdc6 associated with chromatin within 5 min, whereas assembly of MCM proteins was consistently delayed, requiring 10 min (Fig 2). Using sperm or
DNA, we found the kinetics of assembly were indistinguishable. Single-stranded M13 DNA or RNA was unable to bind preinitiation factors in this assay.
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We found that the endogenous cyclin ECdk2 complex bound to chromatin with kinetics similar to ORC and Cdc6 (Fig 2). On chromatin, cyclin E appeared as a doublet, although the fastest migrating, hypophosphorylated form (see Fig 9 B), bound most readily. Quantitative Western blotting indicated that the level of cyclin ECdk2 binding to chromatin was approximately one molecule/origin (see Materials and Methods). This low level of cyclin E was difficult to detect and required exposing the blot shown in Fig 2 overnight. Addition of exogenous cyclin ECdk2 purified from baculovirus increased the total amount of cyclin ECdk2 bound to chromatin (Fig 3 B), suggesting that the number of cyclin ECdk2 chromatin receptors are in excess in HSS extracts. Nonetheless, addition of excess cyclin ECdk2 did not accelerate cyclin E assembly onto chromatin, suggesting that binding of cyclin ECdk2 to chromatin depends on the prior assembly of other factors.
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Assembly of Cyclin ECdk2 onto Chromatin Requires an ATP-dependent Factor in HSS
To determine the requirements for the first phase of cyclin ECdk2 binding to chromatin, we incubated a fixed amount of purified baculovirus cyclin ECdk2 and DNA template with dilutions of HSS, and isolated the assembled chromatin templates. Cyclin E was unable to assemble onto the DNA template in the absence of HSS, but increasing the concentration of HSS caused a linear increase in the amount of cyclin ECdk2 assembled onto chromatin (Fig 3 A), suggesting that extract contains an activity that promotes cyclin E binding to chromatin, which we term a "chromatin receptor."
We determined the biochemical requirements for cyclin ECdk2 recruitment to DNA (Fig 3 B). Heat treatment or ATP depletion of the extract caused a complete loss of cyclin ECdk2 binding to chromatin. ATP depletion (97%) also strongly reduced binding of ORC (Fig 3 B) and Cdc6 (not shown), although a small amount of residual ORC binding to chromatin was observed, likely due to residual ATP-loaded ORC remaining after ATP depletion. Direct binding of yeast ORC to DNA requires ATP (
The ORCCdc6 Preinitiation Complex Acts as a Receptor for Cyclin ECdk2 on Chromatin
To determine whether preinitiation factors facilitated cyclin ECdk2 chromatin recruitment, we depleted ORC, Cdc6, or MCM proteins from HSS before the addition of purified cyclin ECdk2 and DNA. When the assembled chromatin templates were isolated from these samples, we found that a substantial fraction (80%) of the cyclin ECdk2 binding was lost in the absence of ORC and Cdc6, whereas MCM depletion had no significant effect on binding (Fig 3 C). After depletion of ORC or Cdc6,
20% of cyclin ECdk2 did bind to chromatin, even though ORC and Cdc6 depletions appeared quantitative (Fig 3 D, >95%). Therefore, we suspect that the ORCCdc6 complex may not be the only receptor for cyclin ECdk2 on chromatin (see Discussion). Purified Xenopus ORC and recombinant XCdc6 rescued cyclin E binding to chromatin from ORC- or Cdc6-depleted extracts (Fig 3 C). Surprisingly, purified ORC, recombinant Cdc6, and an ATP regenerating system incubated with DNA and purified baculovirus Xcyclin E/XCdk2 could reconstitute a large fraction of cyclin ECdk2 binding to the DNA template (Fig 3 C). If ORC and Cdc6 were not added, no cyclin ECdk2 was recruited to DNA. Thus, in phase one, these two preinitiation factors can function as the cyclin ECdk2 receptor on purified DNA.
Recombinant Cdc6 Binds Directly to the Hypophosphorylated Forms of Cyclin ECdk2 In Vitro
Recent reports have suggested that human Cdc6 binds efficiently to human cyclin A but only weakly to cyclin E (
Although the specific Cdk inhibitors, p21 and p27, could bind all of the various phosphorylated forms of cyclin E, the NH2 terminus of Cdc6 preferentially bound the lower (hypophosphorylated) form (Fig 4), the same form that binds most readily to chromatin. As a control for this type of phosphorylation specificity, we also showed that the cell cycle phosphatase Cdc14, which specifically dephosphorylates mitotically phosphorylated Cdk2 and Cdc2 substrates (Kaiser, B.K., C. Swanson, L. Furstenthal, and P.K. Jackson, manuscript in preparation), binds only the upper hyperphosphorylated forms of cyclin E, likely because Cdc14 binds to the phosphoserine or phosphothreonine moiety of cyclin E before dephosphorylating it. Thus, the interaction of cyclin ECdk2 with Cdc6 appears to be inhibited by cyclin E phosphorylation (see below).
The MRAIL Motif of Cyclin E Is Required to Bind Cdc6, Facilitate Chromatin Recruitment, and Initiate DNA Replication
RXL (Cy) motifs in Cdk substrates and inhibitors are thought to bind to the hydrophobic MRAIL motif in cyclins (
Because the MRAIL domain of cyclin E binds Cdc6, we tested whether the MRAIL mutants of cyclin E stimulate replication. We immunodepleted cyclin E from interphase LSS and added back GST fusions of wild-type or MRAIL mutant Xcyclin E. Although the wild-type cyclin protein (30300 nM) was able to rescue a significant amount of the replication activity in depleted extracts, the mutant protein could not (Fig 5 D). This suggests that the interaction of cyclin E with Cdc6 is essential for DNA replication, although we cannot exclude the possible importance of other substrates of cyclin ECdk2 that require the MRAIL motif. Rescue of the cyclin E depletion with the wild-type GST Xcyclin E protein (45%) was slightly less efficient than rescue with undepleted LSS (59%), which may be due to codepletion of some of the Cdk2 (
Cdc6 Containing Mutations in Its RXL Motifs Is Quantitatively Deficient in Binding to Cyclin E, Phosphorylation by Cyclin ECdk2, and Sustaining DNA Replication
Because the MRAIL motif of cyclin E is required for DNA replication, we tested whether the RXL (Cy) region of Cdc6, which likely binds the cyclin E MRAIL motif, is also important for binding to cyclin E and promoting replication. We constructed GST fusion proteins of XCdc6 containing mutations in one, two, or all three RXL domains, including the first RXL motif (R93, L94, L95), the second (R165, L167), and the third (R258, L260, mutated to alanine). The triple RXL mutant of Cdc6, which had the most dramatic phenotype, was quantitatively impaired in its ability to bind to cyclin E (Fig 6 C) and to be phosphorylated by cyclin ECdk2 in vitro (Fig 6 B), although it retained low levels of both respective activities.
When added to Cdc6-depleted Xenopus extracts, the triple RXL mutant failed to efficiently rescue replication at and below the concentration of XCdc6 in extract (Fig 6 A). Adding the triple mutant protein at high levels (>100 nM) rescued up to 70% as well as the wild-type protein; however, at and below concentrations at which the wild-type protein sustained significant rescuing activity, the mutant was 1.55-fold less effective. The lower the concentration of the mutant, the more deficient it was at rescuing replication compared with wild-type Cdc6. The degree to which the mutant was able to rescue replication correlated completely with its level of binding to cyclin E and its level of phosphorylation by cyclin ECdk2 in vitro. Various combinations of double and single RXL mutants were quantitatively less defective in rescuing replication than the triple mutant; but, the degree of rescue consistently correlated with the number of remaining wild-type RXLs (data not shown). The RXL mutants appear to be otherwise functional, as each bound ORC equivalently to wild-type XCdc6 (data not shown).
Also, we examined a series of Cdc6 NH2-terminal deletion mutants (see Figure S1, available at http://www.jcb.org/cgi/content/full/152/6/1267/DC1). Mutants missing the NH2-terminal 81 or 108 amino acids of Cdc6 bound cyclin E were efficient cyclin ECdk2 substrates in vitro and stimulated DNA replication. However, mutants lacking 178 or 251 NH2-terminal amino acids completely failed to bind cyclin E, be phosphorylated, or stimulate DNA replication. These mutants suggested that additional determinants in the 108178 amino acid sequence (a region that contains only one RXL) are quantitatively important for cyclin E binding and DNA replication. Each of these truncated Cdc6 proteins bound ORC efficiently, suggesting that they were properly folded to retain other activities. These deletion mutants further support the connection between cyclin ECdc6 binding and replication.
Also, we found that an NH2-terminal fragment of XCdc6 (amino acids 1258) containing the cyclin E binding region (Fig 4) inhibited replication at a concentration of 300 nM and completely abrogated replication at
2 µM (data not shown). This is comparable to the concentrations of p21 that inhibits replication and
3.8 times the concentration of endogenous Cdc6 in extract (80 nM;
Cyclin E Accumulation on Chromatin Depends on Polymerase Activity
In a second phase, cyclin E continued to accumulate on chromatin throughout replication (Fig 1 A). Addition of the polymerase inhibitor, aphidicolin, did not effect the initial binding of cyclin E to chromatin but blocked the subsequent accumulation step (Fig 7), indicating that polymerase activity is essential for the accumulation of cyclin ECdk2 on chromatin. Addition of aphidicolin had no effect on the level of Cdc6 (Fig 7) or ORC (not shown) bound to chromatin.
MAP Kinase and Cyclin BCdc2, but Not Plk1, Dissociate Cyclin ECdk2 from Chromatin
To further understand the importance of cyclin ECdk2 recruitment to chromatin, we wanted to define requirements for the mitotic displacement of cyclin E from chromatin (the third phase). This displacement (Fig 1 A and 7) is consistent with previous data showing that Cdc6 is displaced from mitotic chromatin and our data showing that Cdc6 is required for cyclin E binding. However, we also noted that hyperphosphorylated cyclin E, as seen in mitotic extracts (see below), does not bind to Cdc6 (Fig 4).
To determine if any of several essential mitotic kinases were capable of phosphorylating cyclin E and displacing the cyclin ECdk2 complex from chromatin, we treated chromatin assembled in interphase LSS extracts with cyclin BCdc2, MAP kinase, or the polo-like kinase (Plk1) (
The Mitotic Phosphorylation of Cyclin E That Blocks Chromatin Recruitment Can Be Reversed by the Cdc14 Phosphatase
Previously, we had found that during mitosis cyclin ECdk2 is hyperphosphorylated on the cyclin and is approximately threefold increased in activity. This mitotic hyperphosphorylation is inhibited by the Cdk inhibitor p21, indicating that this phosphorylation is Cdk-dependent, likely by one of the mitotic Cdk activities in eggs: cyclin ACdc2, cyclin BCdc2, or cyclin ECdk2 (P.K. Jackson, unpublished data). Cyclin ECdk2 can also autophosphorylate on the cyclin. To correlate the changes in the phosphorylation of cyclin E with mitotic events, we examined the mobility of cyclin E from mitotic or interphase extracts by SDS-PAGE, visualized by Western blotting. Cyclin E was present in at least two forms in interphase extract. Addition of the phosphatase 2A inhibitor and mitotic inducer, okadaic acid, (
To test whether phosphorylation of cyclin E affected chromatin binding, we prepared uniformly autophosphorylated cyclin ECdk2 (Materials and Methods). We observed that hyperphosphorylated cyclin ECdk2 was unable to bind to chromatin, even in the presence of HSS (Fig 9 B). Because Cdc14 can reverse the mitotic phosphorylation of cyclin E in vitro (Fig 8 B) and because Cdc14 is required for mitotic exit in yeast (
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Discussion |
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Cyclin ECdk2 Binds to a Saturable Chromatin Receptor Composed of ORC, Cdc6, and Possibly Other Factor(s)
We have detailed the requirements and cell cycle behavior of the cyclin ECdk2chromatin interaction. A previous study did not see cyclin E associating with chromatin (
There are several reasons why we observe modest levels of cyclin E binding to chromatin in the absence of the nucleus and why we need to add exogenous cyclin ECdk2 to see a strong signal in our HSS chromatin binding assay. Although the major constituents of the cyclin E chromatin receptor, ORC and Cdc6, bind to chromatin with high affinity in membrane-free extracts, we find that nuclear import, or a step subsequent to it, is required for cyclin ECdk2 to bind chromatin efficiently.
ORCCdc6 is one, but may not be the only, receptor for cyclin ECdk2 on chromatin. Our reconstituted chromatin binding reaction allowed us to show that ORC and Cdc6 are required for the first phase of cyclin ECdk2 recruitment to chromatin. Residual cyclin ECdk2 binding to chromatin in the absence of ORC or Cdc6 suggests that there may be other yet unidentified factor(s) that recruit cyclin ECdk2 to chromatin.
By quantitating the amount of cyclin E bound to chromatin during the cell cycle, we gain possible insight into cyclin E's multiple roles in promoting initiation, preventing rereplication, and allowing origin resetting. During the first phase, we see binding of cyclin ECdk2 to chromatin at 100 nM, just above the concentration of cyclin E in interphase cytosol (
60 nM;
1 µM, approximately the concentration of cyclin ECdk2 found in the nucleus soon after nuclear formation. This level of cyclin ECdk2 binding to chromatin corresponds to about one cyclin E molecule per origin during early replication. As replication proceeds, cyclin ECdk2 is deposited on chromatin, dependent on the action of polymerase. We find
510-fold more cyclin E binds by the end of replication (Materials and Methods for calculations). This wide range of cyclin ECdk2 binding, beginning with low binding in phase one before origins have fired, and increasing to high levels throughout phase two as replication proceeds, provides a potential mechanism for the observations that cyclin E both promotes initiation and prevents rereplication. The chromatin substrates of cyclin ECdk2 that become phosphorylated to initiate replication or to block rereplication remain unknown, but ORC and Cdc6 themselves are reasonable candidates (see below).
Cyclin E Uses Its MRAIL Motif to Bind Cdc6 NH2-terminal/RXL Sequences, an Interaction Important for DNA Replication
Our data suggest that the interaction between cyclin E and Cdc6 on chromatin is essential for DNA replication. Work in yeast has also shown that the NH2-terminal 47 amino acids of Cdc6 interact with the Cdk complex that promotes initiation in Saccharomyces cerevisiae, Clb5Cdc28. However, the Cdc6Cdc28 interaction in S. cerevisiae appears to be a complicated one, required at physiological levels of Cdc6, but not when the Cdc6 protein, missing the NH2-terminal amino acid minimal binding domain for cyclinCdc28, is overexpressed (
NH2-terminal deletions of Cdc6, and mutations in the RXL motif cause strong or moderate loss of cyclin ECdk2 binding and a parallel loss in the ability of these Cdc6 variants to stimulate DNA replication. There may be important determinants for cyclin E and Cdc6 to interact in residues 108178, independent of the Cdc6 RXL motifs.
Also, our work suggests a correlation between phosphorylation of Cdc6 and DNA replication. In yeast, phosphorylation of Cdc6 has been shown to play a role in its destruction (
In human USO2 cells, cyclin A, rather than cyclin E, mediates the majority of Cdc6 phosphorylation by Cdks (90% of the Cdk2 is associated with cyclin E (
Mitotic Regulation of the Cyclin ECdk2 Chromatin Association May Be an Important Mechanism in Rereplication Control
We found that mitotic cyclin E hyperphosphorylation apparently causes the cyclin ECdk2 complex to be removed from chromatin. Several arguments suggest that cyclin BCdc2 directly phosphorylates cyclin E in mitosis to cause its displacement from chromatin. First, cyclin E disappears from chromatin after replication is complete (Fig 1 A and 7), when high levels of cyclin BCdc2 activity indicate that the extracts are in mitosis. Second, cyclin E is unable to associate with chromatin assembled in CSF-arrested mitotic extracts in the absence of calcium (Furstenthal, L., and P.K. Jackson, unpublished data) when cyclin B kinase activity is high. Third, the dissociation of cyclin ECdk2 from chromatin assembled in cycling extracts can be blocked by cycloheximide addition, which prevents cyclin B synthesis and entry into mitosis (Fig 1 A). Finally, addition of cyclin BCdc2 to fully assembled interphase chromatin removes cyclin E from the chromatin template (Fig 8 A). The ability of cyclin BCdc2 to phosphorylate recombinant cyclin E in vitro (Fig 8 B) suggests that this effect is direct, rather than an indirect result of inducing mitosis. MAP kinase addition can also dissociate cyclin E from chromatin, although less efficiently than cyclin BCdc2 (Fig 8 A). This result may indicate that MAP kinase is important for keeping cyclin E from rebinding to chromatin in late mitosis, when MAP kinase functions to maintain the mitotic state after Cdc2 inactivation (
Dephosphorylation of cyclin E by Cdc14 reverses the effects of the mitotic kinases and promotes cyclin ECdk2 binding to chromatin. In budding yeast, Cdc14 plays an essential role in the exit from mitosis (
The regulation of cyclin ECdk2 chromatin association by phosphorylation may help explain how cyclin E mediates rereplication control. Oscillations in the level of cyclin ECdk2 are required for Drosophila endocycles, as constitutive expression of cyclin E in Drosophila salivary glands inhibits cell growth and further rounds of DNA replication ( elongation inhibitor, aphidicolin (Fig 7). Our data is thus consistent with cyclin ECdk2 playing a role in both initiation and rereplication control, since it appears to bind additional chromatin receptor(s) as replication progresses and to be stripped from chromatin via phosphorylation by Cdc2 and/or MAP kinase in mitosis. In the next cell cycle, a permissive state for cyclin ECdk2chromatin binding may be reestablished by Cdc14 dephosphorylation of cyclin E upon the exit from mitosis and entry into G1 (Fig 10)
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: CIP, calf intestinal phosphatase; GST, glutathione S-transferase; HSS, high speed supernatant; IP, immunoprecipitation; IVT, in vitro translated; LSS, low speed supernatant; MAP, mitogen-activated protein; MCM, minichromosome maintenance; MOI, multiplicity of infection; MRAIL, Met-Arg-Ala-Ile-Leu; ORC, origin recognition complex; Plk1, polo-like kinase 1; RXL, Arg-X-Leu.
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Acknowledgements |
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We would like to thank Kathy Lacey and Tim Stearns for helpful discussions; Brenda Schulman, Tom Coleman, and Amy Sherman for communicating unpublished results; and Phil Carpenter and Dieter Wolf for critical reading of this manuscript.
This work was supported by National Institutes of Health grant GM54811 and Public Health Services grant CA09302, awarded by the National Cancer Institute.
Submitted: 12 October 2000
Revised: 22 December 2000
Accepted: 23 January 2001
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