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Updated: Jul 17, 2025
Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay
Published on: February 3, 2022
A local ATR-dependent checkpoint pathway is activated by a site-specific replication fork block in human cells
Sana Ahmed-Seghir1, Manisha Jalan1, Helen E Grimsley1
1Department of Radiation Oncology and the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States.
Abstract:
When replication forks encounter DNA lesions that cause polymerase stalling, a checkpoint pathway is activated. The ATR-dependent intra-S checkpoint pathway mediates detection and processing of sites of replication fork stalling to maintain genomic integrity. Several factors involved in the global checkpoint pathway have been identified, but the response to a single replication fork barrier (RFB) is poorly understood. We utilized the Escherichia coli-based Tus-Ter system in human MCF7 cells and showed that the Tus protein binding to TerB sequences creates an efficient site-specific RFB. The single fork RFB was sufficient to activate a local, but not global, ATR-dependent checkpoint response that leads to phosphorylation and accumulation of DNA damage sensor protein γH2AX, confined locally to within a kilobase of the site of stalling. These data support a model of local management of fork stalling, which allows global replication at sites other than the RFB to continue to progress without delay.
Insights
A localized DNA replication checkpoint response manages single replication fork barriers (RFBs). This response, mediated by ATR, prevents global cell cycle arrest, allowing DNA replication to continue elsewhere.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA replication forks can stall when encountering DNA lesions.
- The ATR-dependent intra-S checkpoint pathway is crucial for detecting and processing stalled replication forks to maintain genomic integrity.
- The precise response to a single replication fork barrier (RFB) remains poorly understood.
Purpose of the Study:
- To investigate the cellular response to a single, site-specific replication fork barrier.
- To determine whether a single RFB activates a local or global DNA damage checkpoint response.
Main Methods:
- Utilized the Escherichia coli Tus-Ter system in human MCF7 cells to create a site-specific RFB.
- Monitored the activation of the ATR-dependent checkpoint pathway.
- Assessed the phosphorylation and accumulation of γH2AX at the site of stalling.
Main Results:
- The Tus-Ter system efficiently created a site-specific RFB.
- A single RFB was sufficient to activate a localized ATR-dependent checkpoint response.
- Phosphorylation and accumulation of γH2AX were confined to within a kilobase of the RFB site.
- Global checkpoint activation was not observed.
Conclusions:
- A model of local management of replication fork stalling is supported.
- Localized checkpoint activation allows replication to proceed at other sites without delay.
- This localized response is critical for maintaining genomic integrity during replication stress.
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