An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication
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Updated: Jan 23, 2026
Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification
Published on: November 15, 2010
Tatiana N Moiseeva1, Yandong Yin2, Michael J Calderon3
1Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213.
Abstract:
DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.
Insights
ATR and CHK1 signaling normally limits DNA replication initiation. Inhibiting this pathway causes dormant origins to fire, revealing a mechanism that prevents excessive replication in human cells.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- DNA Replication
Background:
- DNA damage response pathways involving ATR (Ataxia Telangiectasia and Rad3-related) and CHK1 (Checkpoint Kinase 1) are crucial for inhibiting DNA replication and repairing DNA lesions.
- ATR kinase inhibitors are in clinical trials, and studies show they cause excessive origin firing during normal DNA replication, suggesting ATR limits initiation even without damage.
Purpose of the Study:
- To elucidate the mechanism by which ATR and CHK1 signaling limits DNA replication initiation during unperturbed S phase.
- To identify the specific origins affected and the molecular players involved in ATR-mediated regulation of replication initiation.
Main Methods:
- Investigated the role of basal ATR and CHK1 signaling in unperturbed DNA replication.
- Utilized ATR and CHK1 kinase inhibitors to study the effects on origin firing and replication fork stability.
- Examined the phosphorylation status of RIF1 (Replication Initiation Factor 1) and its interaction with PP1 (Protein Phosphatase 1) in response to kinase inhibition.
- Assessed the impact of PP1 activity on CDC7 and CDK2 kinase substrates involved in replicative helicase assembly.
Main Results:
- Unperturbed DNA replication involves low-level ATR and CHK1 signaling that suppresses dormant origin firing.
- Inhibition of ATR/CHK1 signaling leads to the firing of dormant origins throughout S phase.
- ATR/CHK1 inhibition causes CDK1-dependent phosphorylation of RIF1, disrupting its interaction with PP1.
- This disruption releases the inhibition of CDC7 and CDK2, promoting replicative helicase activation.
Conclusions:
- ATR and CHK1 signaling acts as a brake on CDK1 activity during S phase, stabilizing the RIF1-PP1 interaction.
- PP1 dephosphorylates key substrates of CDC7 and CDK2, thereby inhibiting replicative helicase assembly and activation.
- This conserved mechanism ensures limited origin firing during normal DNA replication in human cells.
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