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Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway
1Department of Oncology Research, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Abstract:
In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.
Insights
Checkpoint kinase 2 (Chk2) is activated by DNA damage via ATM kinase and aids in cell cycle arrest and DNA repair. Chk2 also phosphorylates Cdc25C, potentially enhancing cell survival after radiation or topotecan treatment.
Area of Science:
- Cellular biology
- Molecular genetics
- DNA damage response
Background:
- Cell cycle arrest and DNA repair gene transcription are crucial responses to DNA damage.
- ATM (ataxia telangiectasia mutated) kinase and other checkpoint kinases are key regulators in mammalian DNA damage response pathways.
Purpose of the Study:
- To clone and characterize the rat and human homologs of yeast checkpoint kinases, named checkpoint kinase 2 (Chk2).
- To investigate the role of Chk2 in DNA damage and replication stress response pathways, including its relationship with ATM kinase and its function in Cdc25C phosphorylation.
Main Methods:
- Cloning of rat and human Chk2 homologs.
- Yeast complementation studies to assess Chk2 function.
- Analysis of Chk2 activation in response to DNA damage and hydroxyurea (HU) treatment.
- Mass spectrometry to identify Chk2 phosphorylation targets, specifically in Cdc25C.
Main Results:
- Chk2 was cloned and shown to partially rescue the function of a defective checkpoint kinase in yeast.
- Chk2 activation was dependent on ATM kinase following DNA damage but independent of ATM in response to HU-induced replication blocks.
- Mass spectrometry identified Chk2 as a kinase that phosphorylates serine 216 in Cdc25C, a known regulatory site.
Conclusions:
- Chk2 acts as a downstream effector in the ATM-dependent DNA damage checkpoint pathway.
- Chk2 activation may contribute to delaying mitotic entry and improving cell survival following DNA damage induced by gamma-radiation or topotecan.