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Updated: May 29, 2026
Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
Published on: June 9, 2017
Detecting recruitment of DNA damage response factors through the eChIP approach
1Department of Experimental Radiation Oncology, M. D. Anderson Cancer Center, The University of Texas, Houston, TX, USA. ycwang@mdanderson.org
Abstract:
DNA interstrand crosslinks (ICLs) are lesions that covalently link the two strands of DNA. This type of DNA damage represents one of the most complex DNA lesions whose repair mechanisms remain largely unclear. Uncovering proteins involved in the processing of ICLs and understand how they interact with the damaged DNA in vivo is crucial for the understanding of DNA interstrand crosslink repair processes. Moreover, the presence of an ICL during S phase constitutes the most severe blockage to DNA synthesis and results in prolonged stall of replication forks. The mechanisms of resolving a stalled replication fork is poorly understood because proper experimental platforms are lacking. To enable detection of protein recruitment to site-specific ICLs and to ICL-stalled replication forks, we established a novel eChIP (abbreviation for episomal chromatin immunoprecipitation) assay system to study the association of various DNA damage repair proteins with ICL lesions in vivo. This EBV episomal replication-based assay allows detection of protein enrichment at ICLs at the molecular level. Since ICLs cause replication fork blockage in an episomally replicating plasmid, the eChIP approach also allows the study of DNA damage response factor recruitment, such as checkpoint initiation factors, to stalled DNA replication forks. With proper adaptation, the eChIP approach may be employed to study other site-specific DNA lesions such as UV photoproducts and oxidative damage in vivo.
Insights
Researchers developed a novel episomal chromatin immunoprecipitation (eChIP) assay to detect DNA repair proteins at DNA interstrand crosslinks (ICLs) and stalled replication forks in vivo. This method aids in understanding complex DNA repair mechanisms and replication fork resolution.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA interstrand crosslinks (ICLs) are complex DNA lesions that impede DNA replication and whose repair mechanisms are not fully understood.
- Understanding protein interactions with ICLs in vivo is critical for elucidating DNA repair pathways.
- Lack of suitable experimental platforms hinders the study of ICL processing and replication fork stalling.
Purpose of the Study:
- To establish a novel assay for detecting protein recruitment to site-specific ICLs and stalled replication forks in vivo.
- To investigate the association of DNA damage repair proteins with ICL lesions.
- To study the recruitment of DNA damage response factors to stalled replication forks.
Main Methods:
- Development of a novel episomal chromatin immunoprecipitation (eChIP) assay system.
- Utilizing an EBV episomal replication-based system for in vivo studies.
- Detection of protein enrichment at ICLs and stalled replication forks at the molecular level.
Main Results:
- The eChIP assay successfully enabled the detection of protein enrichment at site-specific ICLs in vivo.
- The assay demonstrated the ability to study the recruitment of DNA damage response factors to replication forks stalled by ICLs.
- The eChIP approach provides a molecular-level understanding of protein association with ICLs.
Conclusions:
- The novel eChIP assay is an effective platform for studying DNA interstrand crosslink repair and replication fork dynamics in vivo.
- This method facilitates the investigation of protein interactions with DNA damage and stalled replication forks.
- The eChIP approach has potential applications for studying other DNA lesions like UV photoproducts and oxidative damage.
