DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3^ATR and Cds1^Chk2. However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3^ATR-Cds1^Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1^Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1^Chk2-mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.

DNA复制在复制压力下显着减慢。复制速度的调节在真核生物中是一种保守的反应，在裂殖酵母中，需要检查点激酶 Rad3 ^ATR和 Cds1 ^Chk2。然而，这种检查点监管的潜在机制仍未得到解决。在这里，我们报告 Rad3 ^ATR -Cds1 ^Chk2检查点直接针对复制压力下的 Cdc45-MCM-GINS (CMG) 复制解旋酶。当复制叉停止时，Cds1 ^Chk2激酶直接磷酸化 S275、S322 和 S397 残基上的 Cdc45，从而显着降低 CMG 解旋酶活性。此外，在cds1 ^Chk2突变细胞、CMG 解旋酶和 DNA 聚合酶在物理上是分开的，可能会破坏复制体和折叠复制叉。这项研究表明，S 期内检查点直接调节复制延伸，降低 CMG 解旋酶的持续合成能力，防止 CMG 解旋酶与 DNA 聚合酶脱链，因此有助于保持停滞的复制体和复制叉的完整性。