Limits...
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.

Show MeSH

Related in: MedlinePlus

Excess xHsl7 accelerates entry into mitosis in a nuclear-dependent manner. (A) GST-yeast Hsl7 (lane1) or GST (lane 2) were incubated in Xenopus egg extracts, retrieved on glutathione Sepharose, and immunoblotted with anti-Wee1 antibodies. (B) xHsl7-encoding mRNA or buffer was incubated in cycling extracts with sperm nuclei (5,000/μl) and an ATP-regenerating system. Aliquots were stored at the indicated times and assayed for their ability to phosphorylate histone H1 in the presence of [32P]ATP. Phosphorylated histone was resolved by SDS-PAGE, subjected to autoradiography, and quantified by phosphoimager with the zero time point normalized to 1. Square, buffer; circle, FLAG-xHsl7 mRNA addition. Arrows indicate the time of the nuclear envelope break down and the chromosome condensation as monitored by microscope. (C) Same assay as panel B except that sperm nuclei were absent. (D) In vitro–translated 35S radiolabeled xHsl7 and nucleoplasmin (as an internal control) were added to cycling extracts with or without nuclei present. Samples were withdrawn at the indicated times, resolved by SDS-PAGE, and subjected to autoradiography to monitor xHsl7 stability. Mitosis was observed at 90 min by fluorescence microscopy of extracts containing Hoechst-stained nuclei.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172454&req=5

fig2: Excess xHsl7 accelerates entry into mitosis in a nuclear-dependent manner. (A) GST-yeast Hsl7 (lane1) or GST (lane 2) were incubated in Xenopus egg extracts, retrieved on glutathione Sepharose, and immunoblotted with anti-Wee1 antibodies. (B) xHsl7-encoding mRNA or buffer was incubated in cycling extracts with sperm nuclei (5,000/μl) and an ATP-regenerating system. Aliquots were stored at the indicated times and assayed for their ability to phosphorylate histone H1 in the presence of [32P]ATP. Phosphorylated histone was resolved by SDS-PAGE, subjected to autoradiography, and quantified by phosphoimager with the zero time point normalized to 1. Square, buffer; circle, FLAG-xHsl7 mRNA addition. Arrows indicate the time of the nuclear envelope break down and the chromosome condensation as monitored by microscope. (C) Same assay as panel B except that sperm nuclei were absent. (D) In vitro–translated 35S radiolabeled xHsl7 and nucleoplasmin (as an internal control) were added to cycling extracts with or without nuclei present. Samples were withdrawn at the indicated times, resolved by SDS-PAGE, and subjected to autoradiography to monitor xHsl7 stability. Mitosis was observed at 90 min by fluorescence microscopy of extracts containing Hoechst-stained nuclei.

Mentions: To identify potential Xenopus Hsl7 homologues, we searched the Washington University Xenopus EST database, and identified five Hsl7-related EST clones of various lengths. After DNA sequencing, we identified a full-length Xenopus Hsl7 cDNA (hereafter referred to as xHsl7) bearing 26% identity/44% similarity to yeast Hsl7 and 83% identity/92% similarity to human Hsl7 (known as JBP1 [Janus kinase binding protein 1]/PRMT5; Genbank/EMBL/DDBJ accession no. AY535008) (Fig. 1). We were further encouraged to pursue analysis of this clone as we found that recombinant S. cerevisiae Hsl7 protein readily bound Xenopus Wee1 in egg extracts (Fig. 2 A). This suggested that the Hsl7–Wee1 interaction might be conserved and that the Xenopus protein might also play a role in regulating Wee1 function and mitotic entry.


DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7.

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

Excess xHsl7 accelerates entry into mitosis in a nuclear-dependent manner. (A) GST-yeast Hsl7 (lane1) or GST (lane 2) were incubated in Xenopus egg extracts, retrieved on glutathione Sepharose, and immunoblotted with anti-Wee1 antibodies. (B) xHsl7-encoding mRNA or buffer was incubated in cycling extracts with sperm nuclei (5,000/μl) and an ATP-regenerating system. Aliquots were stored at the indicated times and assayed for their ability to phosphorylate histone H1 in the presence of [32P]ATP. Phosphorylated histone was resolved by SDS-PAGE, subjected to autoradiography, and quantified by phosphoimager with the zero time point normalized to 1. Square, buffer; circle, FLAG-xHsl7 mRNA addition. Arrows indicate the time of the nuclear envelope break down and the chromosome condensation as monitored by microscope. (C) Same assay as panel B except that sperm nuclei were absent. (D) In vitro–translated 35S radiolabeled xHsl7 and nucleoplasmin (as an internal control) were added to cycling extracts with or without nuclei present. Samples were withdrawn at the indicated times, resolved by SDS-PAGE, and subjected to autoradiography to monitor xHsl7 stability. Mitosis was observed at 90 min by fluorescence microscopy of extracts containing Hoechst-stained nuclei.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172454&req=5

fig2: Excess xHsl7 accelerates entry into mitosis in a nuclear-dependent manner. (A) GST-yeast Hsl7 (lane1) or GST (lane 2) were incubated in Xenopus egg extracts, retrieved on glutathione Sepharose, and immunoblotted with anti-Wee1 antibodies. (B) xHsl7-encoding mRNA or buffer was incubated in cycling extracts with sperm nuclei (5,000/μl) and an ATP-regenerating system. Aliquots were stored at the indicated times and assayed for their ability to phosphorylate histone H1 in the presence of [32P]ATP. Phosphorylated histone was resolved by SDS-PAGE, subjected to autoradiography, and quantified by phosphoimager with the zero time point normalized to 1. Square, buffer; circle, FLAG-xHsl7 mRNA addition. Arrows indicate the time of the nuclear envelope break down and the chromosome condensation as monitored by microscope. (C) Same assay as panel B except that sperm nuclei were absent. (D) In vitro–translated 35S radiolabeled xHsl7 and nucleoplasmin (as an internal control) were added to cycling extracts with or without nuclei present. Samples were withdrawn at the indicated times, resolved by SDS-PAGE, and subjected to autoradiography to monitor xHsl7 stability. Mitosis was observed at 90 min by fluorescence microscopy of extracts containing Hoechst-stained nuclei.
Mentions: To identify potential Xenopus Hsl7 homologues, we searched the Washington University Xenopus EST database, and identified five Hsl7-related EST clones of various lengths. After DNA sequencing, we identified a full-length Xenopus Hsl7 cDNA (hereafter referred to as xHsl7) bearing 26% identity/44% similarity to yeast Hsl7 and 83% identity/92% similarity to human Hsl7 (known as JBP1 [Janus kinase binding protein 1]/PRMT5; Genbank/EMBL/DDBJ accession no. AY535008) (Fig. 1). We were further encouraged to pursue analysis of this clone as we found that recombinant S. cerevisiae Hsl7 protein readily bound Xenopus Wee1 in egg extracts (Fig. 2 A). This suggested that the Hsl7–Wee1 interaction might be conserved and that the Xenopus protein might also play a role in regulating Wee1 function and mitotic entry.

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