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Restarting Stalled Replication Forks02:37

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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
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Related Experiment Video

Updated: Feb 13, 2026

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
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A Quantitative DNA Fiber Assay to Monitor Replication Fork Progression, Protection, and Restart.

Debanjali Bhattacharya1, Ganesh Nagaraju1

  • 1Department of Biochemistry, Indian Institute of Science, Bangalore, India.

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|February 12, 2026
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Summary
This summary is machine-generated.

This study introduces a DNA fiber assay for visualizing DNA replication dynamics at single-molecule resolution. This method provides quantitative insights into replication parameters under various cellular stresses, overcoming limitations of previous techniques.

Keywords:
DNA fiber assayFork asymmetryFork protectionFork restartFork speedReplicationReplication stress

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • DNA replication is essential for cell division but faces various stresses.
  • Previous methods for studying replication dynamics had limited spatial resolution and provided average estimates.
  • Understanding replication under physiological and stress conditions is crucial for genome stability.

Purpose of the Study:

  • To describe a novel DNA fiber assay for high-resolution visualization of DNA replication dynamics.
  • To enable quantitative analysis of replication parameters at the single-molecule level.
  • To provide a reproducible method for studying replication under normal and stress conditions.

Main Methods:

  • Incorporation of thymidine analogs (CldU and IdU) into replicating DNA.
  • Stretching and fixation of labeled DNA on glass slides, followed by denaturation.
  • Visualization of labeled nascent DNA using antibodies against CldU/IdU and fluorescence microscopy.

Main Results:

  • The DNA fiber assay allows visualization of individual replicating DNA molecules with single-molecule resolution.
  • The technique is quantitative, high-throughput, and reproducible across different cell lines.
  • It provides insights into DNA synthesis rate, fork protection, restart, and asymmetry under replication stress.

Conclusions:

  • The DNA fiber assay is a powerful and versatile tool for studying DNA replication dynamics.
  • It offers significant advantages over older methods, providing detailed insights into replication mechanisms.
  • This technique is valuable for investigating the impact of genotoxic agents and cellular stresses on genome duplication.