den Elzen Nicole R, O'Connell Matthew J
Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, A'Beckett St., Melbourne, VIC, Australia.
EMBO J. 2004 Feb 25;23(4):908-18. doi: 10.1038/sj.emboj.7600105. Epub 2004 Feb 5.
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.
G2期DNA损伤检查点通过Chk1激酶上调Wee1激酶并下调Cdc25磷酸酶，从而延迟有丝分裂进入，最终导致Cdc2的抑制性磷酸化。虽然检查点激活已被充分了解，但对于检查点如何关闭以允许细胞周期重新进入却知之甚少。为了鉴定检查点释放所需的蛋白质，我们在粟酒裂殖酵母中筛选基因，这些基因在过表达时，会在存在DNA损伤的情况下导致早熟的有丝分裂进入。我们发现I型蛋白磷酸酶Dis2的过表达使粟酒裂殖酵母细胞对DNA损伤敏感，导致异常有丝分裂。Dis2在体内消除Chk1的磷酸化和激活，并在体外使Chk1和磷酸化S345 Chk1肽去磷酸化。dis2Δ细胞具有延长的chk1依赖性停滞，并且下调Chk1活性以进行检查点释放的能力受损。这些效应是DNA损伤检查点特有的，因为Dis2对复制叉停滞的chk1非依赖性反应没有影响。我们提出，Dis2使Chk1失活，从而允许在G2期DNA损伤修复后进入有丝分裂。
