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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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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 nuclei in duplicate. One nuclear sample was extracted with chromatin extraction buffer and respun to isolate chromatin-associated proteins. Cytosolic, nuclear, and chromatin-associated samples were resolved by SDS-PAGE and analyzed by Western blotting with ORC or cyclin E antibodies. Schematics above blots depict the timing of relevant events including, nuclear import (NI), DNA replication (DNA repl), cyclin E association with chromatin (Cyc E on Chrom), and mitosis (M). The indicated samples were supplemented with 10 μM okadaic acid (OA) or 100 μg/ml cycloheximide (CHX) for 120 min. (B) Samples identical to those in A were supplemented with [α-32P]dCTP. At each time point, the reactions were stopped, and the amount of DNA synthesized in duplicate samples was quantitated as detailed in Materials and Methods.
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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 nuclei in duplicate. One nuclear sample was extracted with chromatin extraction buffer and respun to isolate chromatin-associated proteins. Cytosolic, nuclear, and chromatin-associated samples were resolved by SDS-PAGE and analyzed by Western blotting with ORC or cyclin E antibodies. Schematics above blots depict the timing of relevant events including, nuclear import (NI), DNA replication (DNA repl), cyclin E association with chromatin (Cyc E on Chrom), and mitosis (M). The indicated samples were supplemented with 10 μM okadaic acid (OA) or 100 μg/ml cycloheximide (CHX) for 120 min. (B) Samples identical to those in A were supplemented with [α-32P]dCTP. At each time point, the reactions were stopped, and the amount of DNA synthesized in duplicate samples was quantitated as detailed in Materials and Methods.

Mentions: 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 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 (Fig. 1 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 (Fig. 1 A). Cyclin E became detergent-inextractible at the same time that MCMs appear in the detergent-extracted chromatin fractions (not shown).


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)

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 nuclei in duplicate. One nuclear sample was extracted with chromatin extraction buffer and respun to isolate chromatin-associated proteins. Cytosolic, nuclear, and chromatin-associated samples were resolved by SDS-PAGE and analyzed by Western blotting with ORC or cyclin E antibodies. Schematics above blots depict the timing of relevant events including, nuclear import (NI), DNA replication (DNA repl), cyclin E association with chromatin (Cyc E on Chrom), and mitosis (M). The indicated samples were supplemented with 10 μM okadaic acid (OA) or 100 μg/ml cycloheximide (CHX) for 120 min. (B) Samples identical to those in A were supplemented with [α-32P]dCTP. At each time point, the reactions were stopped, and the amount of DNA synthesized in duplicate samples was quantitated as detailed in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199215&req=5

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 nuclei in duplicate. One nuclear sample was extracted with chromatin extraction buffer and respun to isolate chromatin-associated proteins. Cytosolic, nuclear, and chromatin-associated samples were resolved by SDS-PAGE and analyzed by Western blotting with ORC or cyclin E antibodies. Schematics above blots depict the timing of relevant events including, nuclear import (NI), DNA replication (DNA repl), cyclin E association with chromatin (Cyc E on Chrom), and mitosis (M). The indicated samples were supplemented with 10 μM okadaic acid (OA) or 100 μg/ml cycloheximide (CHX) for 120 min. (B) Samples identical to those in A were supplemented with [α-32P]dCTP. At each time point, the reactions were stopped, and the amount of DNA synthesized in duplicate samples was quantitated as detailed in Materials and Methods.
Mentions: 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 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 (Fig. 1 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 (Fig. 1 A). Cyclin E became detergent-inextractible at the same time that MCMs appear in the detergent-extracted chromatin fractions (not shown).

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