Limits...
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.

Show MeSH

Related in: MedlinePlus

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 (lanes 1 and 4), 10 μM okadaic acid (OA; lanes 2 and 6), 1 μM GST–Cdc14 (lanes 3 and 7), or both okadaic acid and Cdc14 (lanes 4 and 8) and incubated at 23°C for 30 min. Reactions were stopped by adding sample buffer, proteins were resolved by SDS-PAGE, and Western blots were performed with cyclin E antibodies to detect the various phosphorylated forms of cyclin E. (B) Baculovirus-expressed Xenopus cyclin E–Cdk2 in an autohyperphosphorylated form was mixed with buffer (lane 2), increasing concentrations of the CIP phosphatase (lanes 3–5), or increasing concentrations of GST–Cdc14 (lanes 6–8) for 30 min. Untreated HSS (lane 1) and treated samples were resolved by SDS-PAGE and analyzed by Western blotting with antibodies to cyclin E (top). The samples in lanes 2–8 were incubated with λ DNA templates and a small amount of HSS (bottom). Assembled chromatin was isolated by sedimentation, and proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti–cyclin E antibodies. The sample in lane 1 is HSS that was not treated (NT).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199215&req=5

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 (lanes 1 and 4), 10 μM okadaic acid (OA; lanes 2 and 6), 1 μM GST–Cdc14 (lanes 3 and 7), or both okadaic acid and Cdc14 (lanes 4 and 8) and incubated at 23°C for 30 min. Reactions were stopped by adding sample buffer, proteins were resolved by SDS-PAGE, and Western blots were performed with cyclin E antibodies to detect the various phosphorylated forms of cyclin E. (B) Baculovirus-expressed Xenopus cyclin E–Cdk2 in an autohyperphosphorylated form was mixed with buffer (lane 2), increasing concentrations of the CIP phosphatase (lanes 3–5), or increasing concentrations of GST–Cdc14 (lanes 6–8) for 30 min. Untreated HSS (lane 1) and treated samples were resolved by SDS-PAGE and analyzed by Western blotting with antibodies to cyclin E (top). The samples in lanes 2–8 were incubated with λ DNA templates and a small amount of HSS (bottom). Assembled chromatin was isolated by sedimentation, and proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti–cyclin E antibodies. The sample in lane 1 is HSS that was not treated (NT).

Mentions: We found that the endogenous cyclin E–Cdk2 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 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 Fig. 2 overnight. Addition of exogenous cyclin E–Cdk2 purified from baculovirus increased the total amount of cyclin E–Cdk2 bound to chromatin (Fig. 3 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.


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)

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 (lanes 1 and 4), 10 μM okadaic acid (OA; lanes 2 and 6), 1 μM GST–Cdc14 (lanes 3 and 7), or both okadaic acid and Cdc14 (lanes 4 and 8) and incubated at 23°C for 30 min. Reactions were stopped by adding sample buffer, proteins were resolved by SDS-PAGE, and Western blots were performed with cyclin E antibodies to detect the various phosphorylated forms of cyclin E. (B) Baculovirus-expressed Xenopus cyclin E–Cdk2 in an autohyperphosphorylated form was mixed with buffer (lane 2), increasing concentrations of the CIP phosphatase (lanes 3–5), or increasing concentrations of GST–Cdc14 (lanes 6–8) for 30 min. Untreated HSS (lane 1) and treated samples were resolved by SDS-PAGE and analyzed by Western blotting with antibodies to cyclin E (top). The samples in lanes 2–8 were incubated with λ DNA templates and a small amount of HSS (bottom). Assembled chromatin was isolated by sedimentation, and proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti–cyclin E antibodies. The sample in lane 1 is HSS that was not treated (NT).
© Copyright Policy
Related In: Results  -  Collection

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

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 (lanes 1 and 4), 10 μM okadaic acid (OA; lanes 2 and 6), 1 μM GST–Cdc14 (lanes 3 and 7), or both okadaic acid and Cdc14 (lanes 4 and 8) and incubated at 23°C for 30 min. Reactions were stopped by adding sample buffer, proteins were resolved by SDS-PAGE, and Western blots were performed with cyclin E antibodies to detect the various phosphorylated forms of cyclin E. (B) Baculovirus-expressed Xenopus cyclin E–Cdk2 in an autohyperphosphorylated form was mixed with buffer (lane 2), increasing concentrations of the CIP phosphatase (lanes 3–5), or increasing concentrations of GST–Cdc14 (lanes 6–8) for 30 min. Untreated HSS (lane 1) and treated samples were resolved by SDS-PAGE and analyzed by Western blotting with antibodies to cyclin E (top). The samples in lanes 2–8 were incubated with λ DNA templates and a small amount of HSS (bottom). Assembled chromatin was isolated by sedimentation, and proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti–cyclin E antibodies. The sample in lane 1 is HSS that was not treated (NT).
Mentions: We found that the endogenous cyclin E–Cdk2 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 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 Fig. 2 overnight. Addition of exogenous cyclin E–Cdk2 purified from baculovirus increased the total amount of cyclin E–Cdk2 bound to chromatin (Fig. 3 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.

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