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

Abstract

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.

The Unstructured C-terminal Tail of Yeast Dpb11 (Human TopBP1) Protein Is Dispensable for DNA Replication and the S Phase Checkpoint but Required for the G2/M Checkpoint

“…The establishment of separation of function mutants of DPB11 was essential to unraveling the contributions made directly by Dpb11 through activation of Mec1 from those made indirectly through its role in the assembly of replication forks. Our studies are generally in agreement with studies of Xenopus TopBP1, in which the replication function of this protein was assigned to the N-terminal half with BRCT domains 1-5, which are homologous to S. cerevisiae BRCT domains 1-4, and the checkpoint function was assigned to the C-terminal half, which contains the ATR-activation domain AAD (25,41).…”

The Rad9-Hus1-Rad1 Checkpoint Clamp Regulates Interaction of TopBP1 with ATR

“…As expected, there was no phosphorylation of Xchk1 in XtopBP1-depleted extracts (9,21,22,33). Consistent with previous reports, we could restore this phosphorylation by adding back wild-type full-length HF-XtopBP1 (9,21,22). However, we could not rescue this defect with ⌬AAD mutant of HF-XtopBP1, which reflected the fact that the AAD was required for the activation of ATR.…”

“…Here we show that this regulated binding involves an N-terminal domain of TopBP1 containing its first two BRCT repeats. This region was not previously known to be important for the checkpoint-regulatory function of TopBP1 (21,22). This segment does not bind directly to ATR-ATRIP, but does interact well with the 9-1-1 complex via the C-terminal region of the Rad9 protein.…”

Reconstitution of a human ATR-mediated checkpoint response to damaged DNA

“…Another PIKK family member, ATR, initiates the DNA damage checkpoint response caused by UV radiation and UV-mimetic agents that produce base damage such as N-acetoxy-2-acetylaminofluorene (N-Aco-AAF). Although this important signal-transduction pathway has been characterized in some detail by using Xenopus egg extracts (9)(10)(11)(12)(13)(14)(15) and in human cell-free systems (16,17), only recently have partial in vitro systems been developed with a subset of either Xenopus (14,15) or yeast (18) checkpoint proteins. Currently, there is no well defined system for ATR-mediated DNA damage checkpoint response in humans.…”