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Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication.

Furstenthal L, Kaiser BK, Swanson C, Jackson PK - J. Cell Biol. (2001)

Bottom Line: In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis.In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin.Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stangford University School of Medicine, Palo Alto, California 94305, USA.

ABSTRACT
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 E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-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 E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted 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 E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed 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 E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.

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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 μg/ml) before isolating chromatin templates by sedimentation and resolving chromatin-associated proteins by SDS-PAGE. Top shows Western blots for cyclin E and Cdc6, which remain bound to chromatin in varying amounts throughout DNA replication (DNA rep). Later time points showed no additional assembly of cyclin E onto chromatin in aphidicolin-treated samples. Bottom shows IP kinase assays of samples identical to those above. Anti–cyclin B antibodies conjugated to protein A–Sepharose beads were used to immunoprecipitate cyclin B, and associated kinase activity was assayed by in vitro phosphorylation of histone H1 in the presence of γ[32P]ATP. The peak in cyclin B kinase activity indicates that the extracts are in mitosis (M).
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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 μg/ml) before isolating chromatin templates by sedimentation and resolving chromatin-associated proteins by SDS-PAGE. Top shows Western blots for cyclin E and Cdc6, which remain bound to chromatin in varying amounts throughout DNA replication (DNA rep). Later time points showed no additional assembly of cyclin E onto chromatin in aphidicolin-treated samples. Bottom shows IP kinase assays of samples identical to those above. Anti–cyclin B antibodies conjugated to protein A–Sepharose beads were used to immunoprecipitate cyclin B, and associated kinase activity was assayed by in vitro phosphorylation of histone H1 in the presence of γ[32P]ATP. The peak in cyclin B kinase activity indicates that the extracts are in mitosis (M).

Mentions: In a second phase, cyclin E continued to accumulate on chromatin throughout replication (Fig. 1 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 (Fig. 7), 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 (Fig. 7) or ORC (not shown) bound to chromatin.


Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication.

Furstenthal L, Kaiser BK, Swanson C, Jackson PK - J. Cell Biol. (2001)

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 μg/ml) before isolating chromatin templates by sedimentation and resolving chromatin-associated proteins by SDS-PAGE. Top shows Western blots for cyclin E and Cdc6, which remain bound to chromatin in varying amounts throughout DNA replication (DNA rep). Later time points showed no additional assembly of cyclin E onto chromatin in aphidicolin-treated samples. Bottom shows IP kinase assays of samples identical to those above. Anti–cyclin B antibodies conjugated to protein A–Sepharose beads were used to immunoprecipitate cyclin B, and associated kinase activity was assayed by in vitro phosphorylation of histone H1 in the presence of γ[32P]ATP. The peak in cyclin B kinase activity indicates that the extracts are in mitosis (M).
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Related In: Results  -  Collection

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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 μg/ml) before isolating chromatin templates by sedimentation and resolving chromatin-associated proteins by SDS-PAGE. Top shows Western blots for cyclin E and Cdc6, which remain bound to chromatin in varying amounts throughout DNA replication (DNA rep). Later time points showed no additional assembly of cyclin E onto chromatin in aphidicolin-treated samples. Bottom shows IP kinase assays of samples identical to those above. Anti–cyclin B antibodies conjugated to protein A–Sepharose beads were used to immunoprecipitate cyclin B, and associated kinase activity was assayed by in vitro phosphorylation of histone H1 in the presence of γ[32P]ATP. The peak in cyclin B kinase activity indicates that the extracts are in mitosis (M).
Mentions: In a second phase, cyclin E continued to accumulate on chromatin throughout replication (Fig. 1 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 (Fig. 7), 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 (Fig. 7) or ORC (not shown) bound to chromatin.

Bottom Line: In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis.In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin.Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stangford University School of Medicine, Palo Alto, California 94305, USA.

ABSTRACT
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 E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-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 E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted 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 E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed 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 E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.

Show MeSH
Related in: MedlinePlus