Site-specific phosphorylation of a checkpoint mediator protein controls its responses to different DNA structures

Abstract

The checkpoint mediator protein Claspin is indispensable for the ATR-dependent phosphorylation of Chk1 in response to stalled DNA replication forks in Xenopus egg extracts. We show that Claspin also participates in the detection of chromosomal double-stranded DNA breaks (DSBs) in this system. Significantly, removal of Claspin from egg extracts only partially abrogates the activation of Chk1 in response to chromatin with DSBs, whereas depletion of both Claspin and BRCA1 completely abolishes this activation. The function of Claspin in this DSB-triggered pathway depends on phosphorylation of T817 and S819 by ATR. Conversely, neither phosphorylation of Claspin on these sites nor the presence of BRCA1 is necessary for activation of Chk1 in response to stalled replication forks. Thus, site-specific phosphorylation of a checkpoint mediator protein is a crucial determinant in the discrimination between various checkpoint-inducing structures. Furthermore, checkpoint mediator proteins exhibit functional overlap that varies depending on the nature of the checkpoint-triggering DNA signal.