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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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RXL mutants of Cdc6 show a quantitative defect in their ability to bind to cyclin E, to get phosphorylated by cyclin E–Cdk2, and to sustain replication in Cdc6-depleted extract. (A) LSS was immunodepleted with affinity-purified XCdc6 antibodies conjugated to protein A–Sepharose beads. Depleted samples were supplemented with sperm DNA, an energy regenerating system, α[32P]dCTP, and 1, 5, 10, 20, 30, or 100 nM of either wild-type GST–XCdc6 (♦) or GST-XCdc6 with all three RXL motifs mutated to AXA (▪) (see Materials and Methods for mutant description). Replication was quantitated as indicated in Materials and Methods and plotted as a percentage of undepleted extract, normalizing to 100% rescue in mock-depleted extracts and setting 0% replication as the amount of background counts incorporated after depletion. (B) Purified GST (lane 1), wild-type GST–XCdc6 (lane 2), or triple RXL mutant GST–XCdc6 (lane 3) was incubated with purified baculovirus-expressed cyclin E–Cdk2 in the presence of γ[32P]ATP. Proteins were resolved by SDS-PAGE, and phosphorylated proteins were visualized by autoradiography. Membrane stained with Ponceau S is shown below as a loading control. (C) Purified GST (lane 1), wild-type GST–XCdc6 (lane 2), or triple RXL mutant GST–XCdc6 (lane 3) was incubated with radiolabeled IVT Xcyclin E. After a 30-min incubation, samples were diluted in IP buffer, and GST proteins were precipitated with glutathione–agarose beads and washed. Beads were resuspended in sample buffer, and associated proteins were resolved by SDS-PAGE and visualized by autoradiography. Membrane stained with Ponceau S is shown below as a loading control.
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Figure 6: RXL mutants of Cdc6 show a quantitative defect in their ability to bind to cyclin E, to get phosphorylated by cyclin E–Cdk2, and to sustain replication in Cdc6-depleted extract. (A) LSS was immunodepleted with affinity-purified XCdc6 antibodies conjugated to protein A–Sepharose beads. Depleted samples were supplemented with sperm DNA, an energy regenerating system, α[32P]dCTP, and 1, 5, 10, 20, 30, or 100 nM of either wild-type GST–XCdc6 (♦) or GST-XCdc6 with all three RXL motifs mutated to AXA (▪) (see Materials and Methods for mutant description). Replication was quantitated as indicated in Materials and Methods and plotted as a percentage of undepleted extract, normalizing to 100% rescue in mock-depleted extracts and setting 0% replication as the amount of background counts incorporated after depletion. (B) Purified GST (lane 1), wild-type GST–XCdc6 (lane 2), or triple RXL mutant GST–XCdc6 (lane 3) was incubated with purified baculovirus-expressed cyclin E–Cdk2 in the presence of γ[32P]ATP. Proteins were resolved by SDS-PAGE, and phosphorylated proteins were visualized by autoradiography. Membrane stained with Ponceau S is shown below as a loading control. (C) Purified GST (lane 1), wild-type GST–XCdc6 (lane 2), or triple RXL mutant GST–XCdc6 (lane 3) was incubated with radiolabeled IVT Xcyclin E. After a 30-min incubation, samples were diluted in IP buffer, and GST proteins were precipitated with glutathione–agarose beads and washed. Beads were resuspended in sample buffer, and associated proteins were resolved by SDS-PAGE and visualized by autoradiography. Membrane stained with Ponceau S is shown below as a loading control.

Mentions: Because the MRAIL motif of cyclin E is required for DNA replication, we tested whether the RXL (Cy) region of Cdc6, which likely binds the cyclin E MRAIL motif, is also important for binding to cyclin E and promoting replication. We constructed GST fusion proteins of XCdc6 containing mutations in one, two, or all three RXL domains, including the first RXL motif (R93, L94, L95), the second (R165, L167), and the third (R258, L260, mutated to alanine). The triple RXL mutant of Cdc6, which had the most dramatic phenotype, was quantitatively impaired in its ability to bind to cyclin E (Fig. 6 C) and to be phosphorylated by cyclin E–Cdk2 in vitro (Fig. 6 B), although it retained low levels of both respective activities.


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)

RXL mutants of Cdc6 show a quantitative defect in their ability to bind to cyclin E, to get phosphorylated by cyclin E–Cdk2, and to sustain replication in Cdc6-depleted extract. (A) LSS was immunodepleted with affinity-purified XCdc6 antibodies conjugated to protein A–Sepharose beads. Depleted samples were supplemented with sperm DNA, an energy regenerating system, α[32P]dCTP, and 1, 5, 10, 20, 30, or 100 nM of either wild-type GST–XCdc6 (♦) or GST-XCdc6 with all three RXL motifs mutated to AXA (▪) (see Materials and Methods for mutant description). Replication was quantitated as indicated in Materials and Methods and plotted as a percentage of undepleted extract, normalizing to 100% rescue in mock-depleted extracts and setting 0% replication as the amount of background counts incorporated after depletion. (B) Purified GST (lane 1), wild-type GST–XCdc6 (lane 2), or triple RXL mutant GST–XCdc6 (lane 3) was incubated with purified baculovirus-expressed cyclin E–Cdk2 in the presence of γ[32P]ATP. Proteins were resolved by SDS-PAGE, and phosphorylated proteins were visualized by autoradiography. Membrane stained with Ponceau S is shown below as a loading control. (C) Purified GST (lane 1), wild-type GST–XCdc6 (lane 2), or triple RXL mutant GST–XCdc6 (lane 3) was incubated with radiolabeled IVT Xcyclin E. After a 30-min incubation, samples were diluted in IP buffer, and GST proteins were precipitated with glutathione–agarose beads and washed. Beads were resuspended in sample buffer, and associated proteins were resolved by SDS-PAGE and visualized by autoradiography. Membrane stained with Ponceau S is shown below as a loading control.
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Figure 6: RXL mutants of Cdc6 show a quantitative defect in their ability to bind to cyclin E, to get phosphorylated by cyclin E–Cdk2, and to sustain replication in Cdc6-depleted extract. (A) LSS was immunodepleted with affinity-purified XCdc6 antibodies conjugated to protein A–Sepharose beads. Depleted samples were supplemented with sperm DNA, an energy regenerating system, α[32P]dCTP, and 1, 5, 10, 20, 30, or 100 nM of either wild-type GST–XCdc6 (♦) or GST-XCdc6 with all three RXL motifs mutated to AXA (▪) (see Materials and Methods for mutant description). Replication was quantitated as indicated in Materials and Methods and plotted as a percentage of undepleted extract, normalizing to 100% rescue in mock-depleted extracts and setting 0% replication as the amount of background counts incorporated after depletion. (B) Purified GST (lane 1), wild-type GST–XCdc6 (lane 2), or triple RXL mutant GST–XCdc6 (lane 3) was incubated with purified baculovirus-expressed cyclin E–Cdk2 in the presence of γ[32P]ATP. Proteins were resolved by SDS-PAGE, and phosphorylated proteins were visualized by autoradiography. Membrane stained with Ponceau S is shown below as a loading control. (C) Purified GST (lane 1), wild-type GST–XCdc6 (lane 2), or triple RXL mutant GST–XCdc6 (lane 3) was incubated with radiolabeled IVT Xcyclin E. After a 30-min incubation, samples were diluted in IP buffer, and GST proteins were precipitated with glutathione–agarose beads and washed. Beads were resuspended in sample buffer, and associated proteins were resolved by SDS-PAGE and visualized by autoradiography. Membrane stained with Ponceau S is shown below as a loading control.
Mentions: Because the MRAIL motif of cyclin E is required for DNA replication, we tested whether the RXL (Cy) region of Cdc6, which likely binds the cyclin E MRAIL motif, is also important for binding to cyclin E and promoting replication. We constructed GST fusion proteins of XCdc6 containing mutations in one, two, or all three RXL domains, including the first RXL motif (R93, L94, L95), the second (R165, L167), and the third (R258, L260, mutated to alanine). The triple RXL mutant of Cdc6, which had the most dramatic phenotype, was quantitatively impaired in its ability to bind to cyclin E (Fig. 6 C) and to be phosphorylated by cyclin E–Cdk2 in vitro (Fig. 6 B), although it retained low levels of both respective activities.

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