Cyclin E–Cdk2 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 ( 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 ( B). Because cyclin E–Cdk2 promotes DNA replication, we tested whether cyclin E–Cdk2 directly interacts with chromatin. We found that cyclin E–Cdk2 associated with chromatin assembled in cycling LSS extracts ( A). In this first phase, cyclin E–Cdk2 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 ( A). Cyclin E became detergent-inextractible at the same time that MCMs appear in the detergent-extracted chromatin fractions (not shown).
Figure 1 Cyclin E associates with chromatin in LSS after nuclear import. (A) Sperm chromatin was assembled in the presence of cycling LSS at 23°C for 0–2 h (time of assembly shown beneath blots) before spinning through a sucrose cushion to isolate (more ...)
In a second phase, cyclin E continued to accumulate on chromatin throughout replication ( A).
In a third phase, chromatin binding of cyclin E–Cdk2 was mitotically regulated. When cyclin B–Cdc2 kinase activity peaked (indicated by the triangle containing an M), cyclin E–Cdk2 was rapidly displaced from chromatin ( 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 ( A) when nuclear envelope breakdown was first initiated. Addition of the phosphatase 2A inhibitor okadaic acid to interphase extracts also induced the mitotic state (Lee et al. 1991
) and displaced both cyclin E and XORC from chromatin. Inhibition of cyclin B synthesis and mitotic entry with the protein synthesis inhibitor cycloheximide blocked cyclin E–Cdk2 displacement. Because DNA replication does not require protein synthesis in LSSs, this indicates that the mitotic state, rather than completion of DNA replication, displaces cyclin E–Cdk2 from chromatin. Cyclin E also appears to be more sensitive to mitotic signals for chromatin displacement than XORC.
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 E–Cdk2 binding to interphase chromatin, we optimized an assay to isolate Xenopus
sperm or λ DNA templates assembled in HSSs of interphase egg extracts (Swedlow and Hirano 1996
). In these extracts, prereplication complexes form, but events after prereplication complex formation are blocked because the extract lacks membranes and cannot assemble nuclei. We find that Xenopus
sperm and λ 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 (). Using sperm or λ DNA, we found the kinetics of assembly were indistinguishable. Single-stranded M13 DNA or RNA was unable to bind preinitiation factors in this assay.
Figure 2 The cyclin E–Cdk2 complex from HSS associates with chromatin with kinetics similar to ORC and Cdc6, but earlier than MCM3. Chromatin was assembled by addition of sperm DNA to HSSs from Xenopus egg extracts, and reactions were stopped at indicated (more ...)
We found that the endogenous cyclin E–Cdk2 complex bound to chromatin with kinetics similar to ORC and Cdc6 (). On chromatin, cyclin E appeared as a doublet, although the fastest migrating, hypophosphorylated form (see B), bound most readily. Quantitative Western blotting indicated that the level of cyclin E–Cdk2 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 overnight. Addition of exogenous cyclin E–Cdk2 purified from baculovirus increased the total amount of cyclin E–Cdk2 bound to chromatin ( B), suggesting that the number of cyclin E–Cdk2 chromatin receptors are in excess in HSS extracts. Nonetheless, addition of excess cyclin E–Cdk2 did not accelerate cyclin E assembly onto chromatin, suggesting that binding of cyclin E–Cdk2 to chromatin depends on the prior assembly of other factors.
Figure 9 Cdc14 reverses the inability of mitotic hyperphosphorylated cyclin E to bind to chromatin. (A) Interphase extract (lanes 1–4) or mitotic extract stabilized by the addition of nondestructible cyclin B (lanes 5–8) was supplemented with buffer (more ...)
Figure 3 To assemble onto chromatin, cyclin E–Cdk2 requires an activity present in HSS that minimally contains ORC and Cdc6. (A) HSS was diluted with XB2 buffer before the addition of λ DNA templates and baculovirus cyclin E–Cdk2 for a (more ...)
Assembly of Cyclin E–Cdk2 onto Chromatin Requires an ATP-dependent Factor in HSS
To determine the requirements for the first phase of cyclin E–Cdk2 binding to chromatin, we incubated a fixed amount of purified baculovirus cyclin E–Cdk2 and λ 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 E–Cdk2 assembled onto chromatin ( 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 E–Cdk2 recruitment to DNA ( B). Heat treatment or ATP depletion of the extract caused a complete loss of cyclin E–Cdk2 binding to chromatin. ATP depletion (97%) also strongly reduced binding of ORC ( 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 (Bell and Stillman 1992
). Addition of excess Mg2+
stimulated the assembly of cyclin E–Cdk2 onto chromatin but not ORC binding ( B). Finally, Cdk activity is not required for recruitment, because addition of the chemical Cdk inhibitor roscovitine had no effect on cyclin E chromatin recruitment (not shown). In contrast, protein Cdk inhibitors, including p21Cip1
(1 μM), did inhibit cyclin E recruitment to chromatin (not shown), likely indicating that they compete with the endogenous receptor protein(s) for binding to cyclin E (see below).
The ORC–Cdc6 Preinitiation Complex Acts as a Receptor for Cyclin E–Cdk2 on Chromatin
To determine whether preinitiation factors facilitated cyclin E–Cdk2 chromatin recruitment, we depleted ORC, Cdc6, or MCM proteins from HSS before the addition of purified cyclin E–Cdk2 and DNA. When the assembled chromatin templates were isolated from these samples, we found that a substantial fraction (~80%) of the cyclin E–Cdk2 binding was lost in the absence of ORC and Cdc6, whereas MCM depletion had no significant effect on binding ( C). After depletion of ORC or Cdc6, ~20% of cyclin E–Cdk2 did bind to chromatin, even though ORC and Cdc6 depletions appeared quantitative ( D, >95%). Therefore, we suspect that the ORC–Cdc6 complex may not be the only receptor for cyclin E–Cdk2 on chromatin (see Discussion). Purified Xenopus ORC and recombinant XCdc6 rescued cyclin E binding to chromatin from ORC- or Cdc6-depleted extracts ( 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 E–Cdk2 binding to the DNA template ( C). If ORC and Cdc6 were not added, no cyclin E–Cdk2 was recruited to DNA. Thus, in phase one, these two preinitiation factors can function as the cyclin E–Cdk2 receptor on purified DNA.
Recombinant Cdc6 Binds Directly to the Hypophosphorylated Forms of Cyclin E–Cdk2 In Vitro
Recent reports have suggested that human Cdc6 binds efficiently to human cyclin A but only weakly to cyclin E (Saha et al. 1998
; Petersen et al. 1999
). However, we find Xenopus
ORC and Cdc6 are sufficient to bind cyclin E–Cdk2 to DNA. Because ORC recruits Cdc6 (Coleman et al. 1996
), we tested whether XCdc6 could bind directly to the Xenopus
cyclin E–Cdk2 complex. When bacterially expressed GST–XCdc6 was incubated together with baculovirus-expressed Xcyclin E–Cdk2, the two proteins efficiently coprecipitated. Addition of an energy regenerating system appeared to stimulate binding but was clearly not essential. Furthermore, the NH2
-terminal half of the Cdc6 protein, which contains all three Cy–RXL motifs (see below), was sufficient for this interaction, whereas the COOH-terminal portion was not ().
Figure 4 Purified cyclin E–Cdk2 binds directly to Cdc6. Baculovirus-expressed cyclin E–Cdk2 was incubated for 30 min with an energy regeneration system and purified GST fusion proteins including GST–p21N1–90 (lane 1), GST–p21C (more ...)
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 (), 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 E–Cdk2 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 (Adams et al. 1996
; Chen et al. 1996
; Russo et al. 1996
; Schulman et al. 1998
). Comparing Cdc6 protein sequences from Xenopus
, human, and mouse, we noted the conservation of two RXL domains (residues 93–95 and 258–260) in the NH2
-terminal half of the protein, surrounded by consensus Cdk phosphorylation sites, with Xenopus
containing a third nonconserved RXL motif (residues 165–167). To test whether the interaction between XCdc6 and Xcyclin E is dependent upon an RXL–MRAIL interaction, we first mutagenized the hydrophobic MRAIL domain of the Xcyclin E protein: amino acids M143
, and W150
, or L186
were mutated to alanine ( A). Unlike the wild-type cyclin E protein, neither mutant bound the inhibitor p21 or the substrate Cdc6 in vitro ( B). Previous studies demonstrated that phosphorylation of RXL-containing cyclin–Cdk substrates require an intact MRAIL sequence in the cyclin, whereas phosphorylation of histone H1 does not (Schulman et al. 1998
). We also found that relative to wild type, our cyclin E mutants phosphorylated histone H1 efficiently but were inefficient at phosphorylating Cdc6 (data not shown). Thus, the mutants retain the activity of properly folded proteins towards substrates, but substrate selectivity is altered. Furthermore, wild-type GST–Xcyclin E could compete with the endogenous cyclin E from HSS for binding to chromatin, but the M143
mutant ( C) and the L186
mutant (not shown) could not. Therefore, an intact MRAIL domain is necessary to compete for the interaction between cyclin E and chromatin.
Figure 5 The MRAIL motif of cyclin E is required for binding of cyclin E to Cdc6, recruitment of cyclin E–Cdk2 to DNA, and replication competence. (A) Schematic of the Xenopus cyclin E protein. The shaded area indicates the cyclin box. Within this region, (more ...)
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 (30–300 nM) was able to rescue a significant amount of the replication activity in depleted extracts, the mutant protein could not ( 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 E–Cdk2 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 (Jackson et al. 1995
), although enough Cdk2 remained to combine with the added cyclin E to rescue a substantial fraction of replication activity.
Cdc6 Containing Mutations in Its RXL Motifs Is Quantitatively Deficient in Binding to Cyclin E, Phosphorylation by Cyclin E–Cdk2, 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 ( C) and to be phosphorylated by cyclin E–Cdk2 in vitro ( B), although it retained low levels of both respective activities.
Figure 6 RXL mutants of Cdc6 show a quantitative defect in their ability to bind to cyclin E, to get phosphorylated by cyclin E–Cdk2, and to sustain replication in Cdc6-depleted extract. (A) LSS was immunodepleted with affinity-purified XCdc6 antibodies (more ...)
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 ( 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 1.5–5-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 E–Cdk2 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 E–Cdk2 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 108–178 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 E–Cdc6 binding and replication.
Also, we found that an NH2
-terminal fragment of XCdc6 (amino acids 1–258) containing the cyclin E binding region () 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; Coleman et al. 1996
). Thus, interfering with the cyclin E–Cdc6 interaction, either by mutation of the RXL motifs in Cdc6, by deletions in the NH2
terminus, or by addition of Cdc6 fragments that bind cyclin E but do not contain the ORC binding region, suppresses replication. Therefore, the first phase of cyclin E recruitment to chromatin by Cdc6 appears to be essential for DNA replication.
Cyclin E Accumulation on Chromatin Depends on Polymerase Activity
In a second phase, cyclin E continued to accumulate on chromatin throughout replication ( A). Addition of the polymerase α inhibitor, aphidicolin, did not effect the initial binding of cyclin E to chromatin but blocked the subsequent accumulation step (), indicating that polymerase activity is essential for the accumulation of cyclin E–Cdk2 on chromatin. Addition of aphidicolin had no effect on the level of Cdc6 () or ORC (not shown) bound to chromatin.
Figure 7 Replication elongation is required for cyclin E accumulation on chromatin. Cycling LSS extracts were incubated with sperm DNA for the indicated times in the absence (lanes 1–6) or the presence (lanes 7 and 8) of aphidicolin (Aphid; 40 (more ...)
MAP Kinase and Cyclin B–Cdc2, but Not Plk1, Dissociate Cyclin E–Cdk2 from Chromatin
To further understand the importance of cyclin E–Cdk2 recruitment to chromatin, we wanted to define requirements for the mitotic displacement of cyclin E from chromatin (the third phase). This displacement ( 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 ().
To determine if any of several essential mitotic kinases were capable of phosphorylating cyclin E and displacing the cyclin E–Cdk2 complex from chromatin, we treated chromatin assembled in interphase LSS extracts with cyclin B–Cdc2, MAP kinase, or the polo-like kinase (Plk1) (Murray and Kirschner 1989
; Lane and Nigg 1996
; Guadagno and Ferrell 1998
) and isolated assembled chromatin. Although treatment with Plk1 had no effect, cyclin B–Cdc2 efficiently removed cyclin E–Cdk2 from chromatin ( A). Addition of MAP kinase could also displace the majority of cyclin E–Cdk2 from chromatin, but less efficiently ( A). Both cyclin B–Cdc2 and MAP kinase phosphorylated purified GST–cyclin E in vitro ( B), suggesting that the effect on cyclin E may be direct. Plk1 also phosphorylated GST–cyclin E in vitro ( B), but the significance of this remains unclear. The Cdc14 phosphatase was capable of reversing the phosphorylation of cyclin E by both Cdc2 and MAP kinase but not by Plk1 ( B), indicating that Plk1 likely phosphorylates cyclin E on different sites from Cdc2 and MAP kinase.
Figure 8 Specific mitotic kinases are capable of phosphorylating cyclin E and displacing cyclin E–Cdk2 from chromatin; Cdc14 can oppose phosphorylation by these kinases. (A) Sperm chromatin assembled in interphase LSS (in the presence of cycloheximide) (more ...)
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 E–Cdk2 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 A–Cdc2, cyclin B–Cdc2, or cyclin E–Cdk2 (P.K. Jackson, unpublished data). Cyclin E–Cdk2 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, (Goris et al. 1989
), resulted in hyperphosphorylation of cyclin E, as did addition of a nondestructible form of cyclin B. This phosphorylation was reversed by the mitotic phosphatase Cdc14 ( A). Cdc14 has been found to be important for the exit from mitosis and appears to function by dephosphorylating substrates of cyclins E, A, and B (Kaiser, B.K., C. Swanson, L. Furstenthal, and P.K. Jackson, manuscript in preparation). In vitro, Cdc14 can directly dephosphorylate cyclin E that has been previously phosphorylated by MAP kinase, cyclin B–Cdc2, or cyclin E–Cdk2 autophosphorylation, but not Plk1 ( B).
To test whether phosphorylation of cyclin E affected chromatin binding, we prepared uniformly autophosphorylated cyclin E–Cdk2 (Materials and Methods). We observed that hyperphosphorylated cyclin E–Cdk2 was unable to bind to chromatin, even in the presence of HSS ( B). Because Cdc14 can reverse the mitotic phosphorylation of cyclin E in vitro ( B) and because Cdc14 is required for mitotic exit in yeast (Wood and Hartwell 1982
; Visintin et al. 1998
), we tested whether Cdc14 would also promote the binding of hyperphosphorylated cyclin E to chromatin. We treated hyperphosphorylated cyclin E–Cdk2 with the Cdc14 phosphatase or with calf intestinal phosphatase (CIP) as a control. Only Cdc14, and not CIP, was able to dephosphorylate cyclin E ( B). The collapse of bands seen in B upon treatment of cyclin E with Cdc14 corresponds to dephosphorylation of cyclin E. When the phosphatase-treated fractions of cyclin E–Cdk2 were tested in the chromatin assembly assay, only the Cdc14-treated dephosphorylated cyclin E bound to chromatin, whereas untreated and CIP-treated fractions did not ( B). Thus, Cdc14 or a similar phosphatase may dephosphorylate mitotic cyclin E–Cdk2 to allow chromatin binding after mitosis, setting up a new round of DNA replication.