Related Experiment Video
Updated: May 31, 2026
08:53
Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
Published on: May 2, 2025
Analysis of protein dynamics at active, stalled, and collapsed replication forks
Bianca M Sirbu1, Frank B Couch, Jordan T Feigerle
1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Genes & Development
|June 21, 2011
Summary
Researchers developed iPOND to track DNA replication and chromatin maturation. This method reveals how cells respond to DNA damage, including histone modifications and checkpoint kinase activation at stalled replication forks.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA replication and chromatin packaging are crucial for genome integrity.
- Tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks in mammalian cells is challenging.
Purpose of the Study:
- To develop a high-resolution technology for analyzing proteins at active and damaged replication forks.
- To define the timing of histone deposition and chromatin maturation during DNA replication.
- To investigate the dynamic protein recruitment and post-translational modifications at stalled replication forks.
Main Methods:
- Developed and utilized a novel technology called iPOND (isolation of proteins on nascent DNA).
- Analyzed protein interactions and modifications at replication forks in mammalian cells.
Main Results:
- Defined the temporal dynamics of histone deposition and chromatin maturation.
- Identified enrichment of Class 1 histone deacetylases at replisomes, removing predeposition marks on histone H4.
- Observed that chromatin maturation can occur independently of replisome movement.
- Characterized changes in protein recruitment and phosphorylation at stalled forks, including checkpoint kinase-catalyzed H2AX phosphorylation.
- Demonstrated a switch in DNA damage response at persistently stalled forks involving MRE11-dependent RAD51 assembly.
Conclusions:
- iPOND is a valuable methodology for studying DNA replication and chromatin maturation.
- Revealed dynamic protein recruitment and post-translational modifications at active and damaged replication forks.
- Provided insights into the cellular response to replication stress and DNA damage.
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