The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activation of Chk1.

ATR-dependent activation of the kinase Chk1 is the initial step in signal transduction in the DNA replication checkpoint, which allows a cell to enter mitosis only after the completion of DNA replication. TopBP1-related proteins in higher eukaryotes are implicated in the replication checkpoint, but their exact role remains elusive because of their requirements for replication initiation. Here we report that the initiation function of Xenopus Cut5/TopBP1 could be entirely separated from its checkpoint function: the N-terminal half fragment, a region of Cut5 conserved through evolution, is sufficient for initiation, but is incapable of activating the checkpoint; the C-terminal half fragment, which is unique in metazoan species, is by itself capable of activating the checkpoint response without initiating replication. Upon the activation of Chk1, the Ser1131 within the C-terminal region of Cut5 is phosphorylated, and this phosphorylation is critical for the checkpoint response. Furthermore, Cut5 directly stimulated Chk1 phosphorylation in the in vitro kinase assay reconstituted with recombinant proteins and ATR immunoprecipitated from extracts. On the basis of replication protein A (RPA)-dependent loading of Cut5 on to replicating and replication-arrested chromatin, we propose that Cut5 plays a crucial role in the initial amplification step of the ATR-Chk1 signaling pathway at the stalled replication fork.