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DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7.

Yamada A, Duffy B, Perry JA, Kornbluth S - J. Cell Biol. (2004)

Bottom Line: Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction.Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase.These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

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Excess Hsl7 overrides the DNA replication checkpoint. (A) Xenopus Hsl7 or β-globin mRNA was incubated in cycling extracts in the presence of aphidicolin (200 μg/ml). Cell cycle progression was monitored by assessing histone H1 kinase activity. Square, β-globin mRNA; circle, FLAG-xHsl7 mRNA. An arrow indicates the time of the nuclear envelope break down and the chromosome condensation as monitored by a microscope. (B) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2,3, 4) were incubated in cycling extracts containing no sperm (lane 2) or sperm chromatin DNA (lanes 1, 3, and 4) in the absence (lanes 1–3) or presence (lane 4) of aphidicolin (200 μg/ml). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (C) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2–4) were incubated in cycling extracts containing sperm chromatin DNA in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of aphidicolin (200 μg/ml) and inactive Plx1 (lane 3) or constitutively active Plx1 (T201D; lane 4). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting.
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fig7: Excess Hsl7 overrides the DNA replication checkpoint. (A) Xenopus Hsl7 or β-globin mRNA was incubated in cycling extracts in the presence of aphidicolin (200 μg/ml). Cell cycle progression was monitored by assessing histone H1 kinase activity. Square, β-globin mRNA; circle, FLAG-xHsl7 mRNA. An arrow indicates the time of the nuclear envelope break down and the chromosome condensation as monitored by a microscope. (B) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2,3, 4) were incubated in cycling extracts containing no sperm (lane 2) or sperm chromatin DNA (lanes 1, 3, and 4) in the absence (lanes 1–3) or presence (lane 4) of aphidicolin (200 μg/ml). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (C) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2–4) were incubated in cycling extracts containing sperm chromatin DNA in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of aphidicolin (200 μg/ml) and inactive Plx1 (lane 3) or constitutively active Plx1 (T201D; lane 4). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting.

Mentions: In S. cerevisiae, inhibition of budding or actin depolymerization triggers the morphogenesis checkpoint to prevent Hsl7-stimulated Swe1 degradation (McMillan et al., 1999). Accordingly, overexpression of Hsl7 will override the morphogenesis checkpoint. Using similar logic, it was attractive to speculate that overexpression of xHsl7 might override the DNA replication checkpoint as this has been reported to promote stabilization of nuclear Wee1. To test this, we added excess xHsl7 mRNA to cycling extracts supplemented with nuclei and the DNA polymerase inhibitor, aphidicolin, and monitored activation of Cdc2–cyclin B1-catalyzed histone H1 kinase activity. Remarkably, excess xHsl7 abrogated G2/M arrest by the checkpoint, allowing the efficient activation of Cdc2–cyclin B and entry into mitosis even when DNA synthesis was inhibited (Fig. 7 A).


DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7.

Yamada A, Duffy B, Perry JA, Kornbluth S - J. Cell Biol. (2004)

Excess Hsl7 overrides the DNA replication checkpoint. (A) Xenopus Hsl7 or β-globin mRNA was incubated in cycling extracts in the presence of aphidicolin (200 μg/ml). Cell cycle progression was monitored by assessing histone H1 kinase activity. Square, β-globin mRNA; circle, FLAG-xHsl7 mRNA. An arrow indicates the time of the nuclear envelope break down and the chromosome condensation as monitored by a microscope. (B) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2,3, 4) were incubated in cycling extracts containing no sperm (lane 2) or sperm chromatin DNA (lanes 1, 3, and 4) in the absence (lanes 1–3) or presence (lane 4) of aphidicolin (200 μg/ml). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (C) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2–4) were incubated in cycling extracts containing sperm chromatin DNA in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of aphidicolin (200 μg/ml) and inactive Plx1 (lane 3) or constitutively active Plx1 (T201D; lane 4). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting.
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Related In: Results  -  Collection

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fig7: Excess Hsl7 overrides the DNA replication checkpoint. (A) Xenopus Hsl7 or β-globin mRNA was incubated in cycling extracts in the presence of aphidicolin (200 μg/ml). Cell cycle progression was monitored by assessing histone H1 kinase activity. Square, β-globin mRNA; circle, FLAG-xHsl7 mRNA. An arrow indicates the time of the nuclear envelope break down and the chromosome condensation as monitored by a microscope. (B) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2,3, 4) were incubated in cycling extracts containing no sperm (lane 2) or sperm chromatin DNA (lanes 1, 3, and 4) in the absence (lanes 1–3) or presence (lane 4) of aphidicolin (200 μg/ml). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (C) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2–4) were incubated in cycling extracts containing sperm chromatin DNA in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of aphidicolin (200 μg/ml) and inactive Plx1 (lane 3) or constitutively active Plx1 (T201D; lane 4). After a 60-min incubation, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting.
Mentions: In S. cerevisiae, inhibition of budding or actin depolymerization triggers the morphogenesis checkpoint to prevent Hsl7-stimulated Swe1 degradation (McMillan et al., 1999). Accordingly, overexpression of Hsl7 will override the morphogenesis checkpoint. Using similar logic, it was attractive to speculate that overexpression of xHsl7 might override the DNA replication checkpoint as this has been reported to promote stabilization of nuclear Wee1. To test this, we added excess xHsl7 mRNA to cycling extracts supplemented with nuclei and the DNA polymerase inhibitor, aphidicolin, and monitored activation of Cdc2–cyclin B1-catalyzed histone H1 kinase activity. Remarkably, excess xHsl7 abrogated G2/M arrest by the checkpoint, allowing the efficient activation of Cdc2–cyclin B and entry into mitosis even when DNA synthesis was inhibited (Fig. 7 A).

Bottom Line: Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction.Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase.These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

Show MeSH
Related in: MedlinePlus