EMBO J. 2004 Feb 25;23(4):908-18. Epub 2004 Feb 5.

Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1.

Author information

1: Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, A'Beckett St., Melbourne, VIC, Australia.

Abstract

The G2 DNA damage checkpoint delays mitotic entry via the upregulation of Wee1 kinase and the downregulation of Cdc25 phosphatase by Chk1 kinase, and resultant inhibitory phosphorylation of Cdc2. While checkpoint activation is well understood, little is known about how the checkpoint is switched off to allow cell cycle re-entry. To identify proteins required for checkpoint release, we screened for genes in Schizosaccharomyces pombe that, when overexpressed, result in precocious mitotic entry in the presence of DNA damage. We show that overexpression of the type I protein phosphatase Dis2 sensitises S. pombe cells to DNA damage, causing aberrant mitoses. Dis2 abrogates Chk1 phosphorylation and activation in vivo, and dephosphorylates Chk1 and a phospho-S345 Chk1 peptide in vitro. dis2Delta cells have a prolonged chk1-dependent arrest and a compromised ability to downregulate Chk1 activity for checkpoint release. These effects are specific for the DNA damage checkpoint, because Dis2 has no effect on the chk1-independent response to stalled replication forks. We propose that inactivation of Chk1 by Dis2 allows mitotic entry following repair of DNA damage in the G2-phase.

PMID:: 14765108
PMCID:: PMC381011
DOI:: 10.1038/sj.emboj.7600105

[Indexed for MEDLINE]

Free PMC Article

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Figure 1

Dis2 overexpression sensitises cells to DNA damage. (A) Wild-type cells transformed with vector or nmt1::dis2 plasmid were grown in the absence of thiamine for 16 h at 30°C and, together with checkpoint mutant strains rad3-136 and chk1Δ, serial dilutions were spotted onto minimal medium agar containing no drug, 0.013% MMS or 62 ng/ml 4-NQO. (B) DAPI-stained cells of strains grown as described in (A), and treated with 0.01% MMS for 16 h. (C) Wild-type cells transformed with vector (○) or nmt1::dis2 plasmid (•), together with chk1Δ (□) and rad3-136 (▵), were treated with the indicated doses of UV-C and ionising radiation, and per cent survival compared with unirradiated controls was determined. (D) Wild-type and t.s. mutant strains transformed with vector (○) or nmt1::dis2 (•), or crossed with chk1Δ (□), were grown in the absence of thiamine for 24 h at 25°C, then shifted to the restrictive temperature of 36°C, and the per cent of cells passing mitosis or with aberrant mitoses was followed over time.

Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1