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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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To assemble onto chromatin, cyclin E–Cdk2 requires an activity present in HSS that minimally contains ORC and Cdc6. (A) HSS was diluted with XB2 buffer before the addition of λ DNA templates and baculovirus cyclin E–Cdk2 for a 30-min incubation. Assembled chromatin was isolated and analyzed as in Fig. 1. Lane 1, no DNA; lanes 2–6, DNA templates assembled in HSS that was undiluted, or diluted 1:1, 1:3, 1:7, or 1:11 with XB2. (B) HSS was either left untreated (lanes 1–3), heat treated (lane 4), ATP depleted (lane 5), or supplemented with 10 mM MgCl2 (lane 6) before the addition of λ DNA templates (lanes 2–6). Purified baculovirus-expressed cyclin E–Cdk2 was also added to samples in lanes 3–6. Assembled chromatin was isolated and analyzed as above. (C) Individual aliquots of HSS were immunodepleted with antibodies specific to XORC2 (lanes 3 and 6), XCdc6 (lanes 4 and 7), XMCM3 (lane 5), or with beads alone (lane 2). Specific samples were supplemented with purified XORC complex (lanes 6 and 8) or baculovirus-expressed XCdc6 (lanes 7 and 8). All samples included baculovirus-expressed Xcyclin E–Cdk2 and an energy regenerating system. Depleted samples with and without additions were incubated with λ DNA for 30 min, sedimented through a sucrose cushion, and resolved by SDS-PAGE. (D) Western blots of depleted HSS used for assembling chromatin in C. Lane 1, mock depleted; lane 2, ORC2 depleted; lane 3, Cdc6 depleted; lane 4, MCM3 depleted.
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Figure 3: To assemble onto chromatin, cyclin E–Cdk2 requires an activity present in HSS that minimally contains ORC and Cdc6. (A) HSS was diluted with XB2 buffer before the addition of λ DNA templates and baculovirus cyclin E–Cdk2 for a 30-min incubation. Assembled chromatin was isolated and analyzed as in Fig. 1. Lane 1, no DNA; lanes 2–6, DNA templates assembled in HSS that was undiluted, or diluted 1:1, 1:3, 1:7, or 1:11 with XB2. (B) HSS was either left untreated (lanes 1–3), heat treated (lane 4), ATP depleted (lane 5), or supplemented with 10 mM MgCl2 (lane 6) before the addition of λ DNA templates (lanes 2–6). Purified baculovirus-expressed cyclin E–Cdk2 was also added to samples in lanes 3–6. Assembled chromatin was isolated and analyzed as above. (C) Individual aliquots of HSS were immunodepleted with antibodies specific to XORC2 (lanes 3 and 6), XCdc6 (lanes 4 and 7), XMCM3 (lane 5), or with beads alone (lane 2). Specific samples were supplemented with purified XORC complex (lanes 6 and 8) or baculovirus-expressed XCdc6 (lanes 7 and 8). All samples included baculovirus-expressed Xcyclin E–Cdk2 and an energy regenerating system. Depleted samples with and without additions were incubated with λ DNA for 30 min, sedimented through a sucrose cushion, and resolved by SDS-PAGE. (D) Western blots of depleted HSS used for assembling chromatin in C. Lane 1, mock depleted; lane 2, ORC2 depleted; lane 3, Cdc6 depleted; lane 4, MCM3 depleted.

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)

To assemble onto chromatin, cyclin E–Cdk2 requires an activity present in HSS that minimally contains ORC and Cdc6. (A) HSS was diluted with XB2 buffer before the addition of λ DNA templates and baculovirus cyclin E–Cdk2 for a 30-min incubation. Assembled chromatin was isolated and analyzed as in Fig. 1. Lane 1, no DNA; lanes 2–6, DNA templates assembled in HSS that was undiluted, or diluted 1:1, 1:3, 1:7, or 1:11 with XB2. (B) HSS was either left untreated (lanes 1–3), heat treated (lane 4), ATP depleted (lane 5), or supplemented with 10 mM MgCl2 (lane 6) before the addition of λ DNA templates (lanes 2–6). Purified baculovirus-expressed cyclin E–Cdk2 was also added to samples in lanes 3–6. Assembled chromatin was isolated and analyzed as above. (C) Individual aliquots of HSS were immunodepleted with antibodies specific to XORC2 (lanes 3 and 6), XCdc6 (lanes 4 and 7), XMCM3 (lane 5), or with beads alone (lane 2). Specific samples were supplemented with purified XORC complex (lanes 6 and 8) or baculovirus-expressed XCdc6 (lanes 7 and 8). All samples included baculovirus-expressed Xcyclin E–Cdk2 and an energy regenerating system. Depleted samples with and without additions were incubated with λ DNA for 30 min, sedimented through a sucrose cushion, and resolved by SDS-PAGE. (D) Western blots of depleted HSS used for assembling chromatin in C. Lane 1, mock depleted; lane 2, ORC2 depleted; lane 3, Cdc6 depleted; lane 4, MCM3 depleted.
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Figure 3: To assemble onto chromatin, cyclin E–Cdk2 requires an activity present in HSS that minimally contains ORC and Cdc6. (A) HSS was diluted with XB2 buffer before the addition of λ DNA templates and baculovirus cyclin E–Cdk2 for a 30-min incubation. Assembled chromatin was isolated and analyzed as in Fig. 1. Lane 1, no DNA; lanes 2–6, DNA templates assembled in HSS that was undiluted, or diluted 1:1, 1:3, 1:7, or 1:11 with XB2. (B) HSS was either left untreated (lanes 1–3), heat treated (lane 4), ATP depleted (lane 5), or supplemented with 10 mM MgCl2 (lane 6) before the addition of λ DNA templates (lanes 2–6). Purified baculovirus-expressed cyclin E–Cdk2 was also added to samples in lanes 3–6. Assembled chromatin was isolated and analyzed as above. (C) Individual aliquots of HSS were immunodepleted with antibodies specific to XORC2 (lanes 3 and 6), XCdc6 (lanes 4 and 7), XMCM3 (lane 5), or with beads alone (lane 2). Specific samples were supplemented with purified XORC complex (lanes 6 and 8) or baculovirus-expressed XCdc6 (lanes 7 and 8). All samples included baculovirus-expressed Xcyclin E–Cdk2 and an energy regenerating system. Depleted samples with and without additions were incubated with λ DNA for 30 min, sedimented through a sucrose cushion, and resolved by SDS-PAGE. (D) Western blots of depleted HSS used for assembling chromatin in C. Lane 1, mock depleted; lane 2, ORC2 depleted; lane 3, Cdc6 depleted; lane 4, MCM3 depleted.
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