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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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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–p21C87–164 (lane 2), GST–p27 (lane 3), GST–Cdc6N2–168 (lane 4), GST–Cdc6C169–554 (lane 5), GST–XORC1 (lane 6), or GST–hCdc14 (lane 7). Reactions were diluted in IP buffer and bound to glutathione–agarose beads. Beads were washed and resolved by SDS-PAGE, and cyclin E was visualized by Western blotting.
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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–p21C87–164 (lane 2), GST–p27 (lane 3), GST–Cdc6N2–168 (lane 4), GST–Cdc6C169–554 (lane 5), GST–XORC1 (lane 6), or GST–hCdc14 (lane 7). Reactions were diluted in IP buffer and bound to glutathione–agarose beads. Beads were washed and resolved by SDS-PAGE, and cyclin E was visualized by Western blotting.

Mentions: 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 (Fig. 4).


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)

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–p21C87–164 (lane 2), GST–p27 (lane 3), GST–Cdc6N2–168 (lane 4), GST–Cdc6C169–554 (lane 5), GST–XORC1 (lane 6), or GST–hCdc14 (lane 7). Reactions were diluted in IP buffer and bound to glutathione–agarose beads. Beads were washed and resolved by SDS-PAGE, and cyclin E was visualized by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

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

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–p21C87–164 (lane 2), GST–p27 (lane 3), GST–Cdc6N2–168 (lane 4), GST–Cdc6C169–554 (lane 5), GST–XORC1 (lane 6), or GST–hCdc14 (lane 7). Reactions were diluted in IP buffer and bound to glutathione–agarose beads. Beads were washed and resolved by SDS-PAGE, and cyclin E was visualized by Western blotting.
Mentions: 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 (Fig. 4).

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