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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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Model of the cell cycle–regulated association of cyclin E–Cdk2 with chromatin and its effects on DNA replication and rereplication control. In a first phase, cyclin E–Cdk2 is recruited to origins of DNA replication by ORC, Cdc6, and possibly an unknown factor (X?). In this conformation, with MCMs bound, DNA replication is initiated. In a second phase, dependent on the progression of replication forks, multiple molecules of cyclin E accumulate on chromatin, blocking re-replication. In a final phase, cyclin E is hyperphosphorylated by cyclin B–Cdc2 and stripped from chromatin in mitosis. Rebinding of cyclin E to chromatin in interphase is possible only after dephosphorylation by Cdc14 or a related phosphatase (Discussion).
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Figure 10: Model of the cell cycle–regulated association of cyclin E–Cdk2 with chromatin and its effects on DNA replication and rereplication control. In a first phase, cyclin E–Cdk2 is recruited to origins of DNA replication by ORC, Cdc6, and possibly an unknown factor (X?). In this conformation, with MCMs bound, DNA replication is initiated. In a second phase, dependent on the progression of replication forks, multiple molecules of cyclin E accumulate on chromatin, blocking re-replication. In a final phase, cyclin E is hyperphosphorylated by cyclin B–Cdc2 and stripped from chromatin in mitosis. Rebinding of cyclin E to chromatin in interphase is possible only after dephosphorylation by Cdc14 or a related phosphatase (Discussion).

Mentions: The regulation of cyclin E–Cdk2 chromatin association by phosphorylation may help explain how cyclin E mediates rereplication control. Oscillations in the level of cyclin E–Cdk2 are required for Drosophila endocycles, as constitutive expression of cyclin E in Drosophila salivary glands inhibits cell growth and further rounds of DNA replication (Follette et al. 1998). A similar phenomenon was reported in Xenopus extracts, which are unable to replicate in the presence of high levels of cyclin E–Cdk2 (Hua et al. 1997). We show that, in phase two, cyclin E accumulates on chromatin as replication progresses (Fig. 1 A) and that chromatin accumulation of cyclin E can be blocked at stage one levels by addition of the polymerase α elongation inhibitor, aphidicolin (Fig. 7). Our data is thus consistent with cyclin E–Cdk2 playing a role in both initiation and rereplication control, since it appears to bind additional chromatin receptor(s) as replication progresses and to be stripped from chromatin via phosphorylation by Cdc2 and/or MAP kinase in mitosis. In the next cell cycle, a permissive state for cyclin E–Cdk2–chromatin binding may be reestablished by Cdc14 dephosphorylation of cyclin E upon the exit from mitosis and entry into G1 (Fig. 10)


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)

Model of the cell cycle–regulated association of cyclin E–Cdk2 with chromatin and its effects on DNA replication and rereplication control. In a first phase, cyclin E–Cdk2 is recruited to origins of DNA replication by ORC, Cdc6, and possibly an unknown factor (X?). In this conformation, with MCMs bound, DNA replication is initiated. In a second phase, dependent on the progression of replication forks, multiple molecules of cyclin E accumulate on chromatin, blocking re-replication. In a final phase, cyclin E is hyperphosphorylated by cyclin B–Cdc2 and stripped from chromatin in mitosis. Rebinding of cyclin E to chromatin in interphase is possible only after dephosphorylation by Cdc14 or a related phosphatase (Discussion).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Model of the cell cycle–regulated association of cyclin E–Cdk2 with chromatin and its effects on DNA replication and rereplication control. In a first phase, cyclin E–Cdk2 is recruited to origins of DNA replication by ORC, Cdc6, and possibly an unknown factor (X?). In this conformation, with MCMs bound, DNA replication is initiated. In a second phase, dependent on the progression of replication forks, multiple molecules of cyclin E accumulate on chromatin, blocking re-replication. In a final phase, cyclin E is hyperphosphorylated by cyclin B–Cdc2 and stripped from chromatin in mitosis. Rebinding of cyclin E to chromatin in interphase is possible only after dephosphorylation by Cdc14 or a related phosphatase (Discussion).
Mentions: The regulation of cyclin E–Cdk2 chromatin association by phosphorylation may help explain how cyclin E mediates rereplication control. Oscillations in the level of cyclin E–Cdk2 are required for Drosophila endocycles, as constitutive expression of cyclin E in Drosophila salivary glands inhibits cell growth and further rounds of DNA replication (Follette et al. 1998). A similar phenomenon was reported in Xenopus extracts, which are unable to replicate in the presence of high levels of cyclin E–Cdk2 (Hua et al. 1997). We show that, in phase two, cyclin E accumulates on chromatin as replication progresses (Fig. 1 A) and that chromatin accumulation of cyclin E can be blocked at stage one levels by addition of the polymerase α elongation inhibitor, aphidicolin (Fig. 7). Our data is thus consistent with cyclin E–Cdk2 playing a role in both initiation and rereplication control, since it appears to bind additional chromatin receptor(s) as replication progresses and to be stripped from chromatin via phosphorylation by Cdc2 and/or MAP kinase in mitosis. In the next cell cycle, a permissive state for cyclin E–Cdk2–chromatin binding may be reestablished by Cdc14 dephosphorylation of cyclin E upon the exit from mitosis and entry into G1 (Fig. 10)

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