Sci. STKE, 18 April 2006
CHECKPOINT SIGNALING Discriminating DNA Structures
Because DNA fidelity is crucial for cell survival and physiology, there are elaborate mechanisms to ensure that DNA is replicated properly and that DNA damage, such as double-stranded breaks (DSBs), is repaired before completion of the cell cycle. The repair processes are regulated by signaling through checkpoint pathways that involve kinases and various adaptors, as well as the enzymes and molecules that repair the damaged DNA. Yoo et al. used Xenopus egg extracts and sperm chromatin to study the role of the adaptors claspin and BRCA1 and the kinases ATR and Chk1 in checkpoint responses to DSBs and stalled DNA replication forks. The interaction between claspin and DNA was increased by the addition of the restriction enzyme Eco RI to cause DSBs or aphidicolin to stall DNA replication. Claspin mediates the interaction with and phosphorylation of Chk1 by ATR. The interaction between claspin and Chk1 requires that claspin is also phosphorylated on Ser864 and S895 in a manner dependent on ATR, although ATR is not the kinase responsible for directly phosphorylating these residues of claspin. Both claspin and BRCA1 appeared to contribute to Chk1 activation in response to EcoRI-created DSBs, because only when both adaptors were removed from the extracts by immunodepletion was Chk1 phosphorylation blocked. Immunodepletion of claspin alone blocked phosphorylation of Chk1 in response to aphidicolin-induced stalled DNA replication forks. Yoo et al. mutated 12 potential phosphorylation sites in claspin that matched the consensus motif for ATR-mediated phosphorylation and found that activation of Chk1 in response to aphidicolin-induced stalled replication forks was unaffected, whereas the activation of Chk1 in response to DSBs was decreased. Mutation of Thr817 and Ser819, but not either residue alone, abolished Chk1 activation in response to DSBs. Furthermore, phosphorylation of the wild-type protein on Thr817 and Ser819 was detected in the egg extracts, and these residues were phosphorylated in extracts containing DNA with DSBs or stalled replication forks. In vitro, ATR phosphorylated a claspin fragment containing Thr817 and Ser819, and immunodepletion of ATR from the extracts blocked phosphorylation of Ser819 (based on loss of immunoreactivity of claspin with phosphorylation-dependent antibodies against this site). Although Thr817 and Ser819 are phosphorylated in response to both kinds of DNA structures (stalled forks and DSBs), this phosphorylation appears only to be critical for mediating Chk1 activation in response to DSBs.
H. Y. Yoo, S.-Y. Jeong, W. G. Dunphy, Site-specific phosphorylation of a checkpoint mediator protein controls its responses to different DNA structures. Genes Dev. 20, 772-783 (2006). [Abstract] [Full Text]
Citation: Discriminating DNA Structures. Sci. STKE 2006, tw131 (2006).
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