Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

5.8K
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,...
5.8K
The DNA Replication Fork01:02

The DNA Replication Fork

35.9K
An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...
35.9K
The Replisome03:01

The Replisome

33.4K
DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
33.4K
DNA Damage can Stall the Cell Cycle02:37

DNA Damage can Stall the Cell Cycle

9.1K
In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
9.1K
Replication in Eukaryotes02:31

Replication in Eukaryotes

170.7K
Overview
170.7K
Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

14.1K
For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
14.1K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

mRNA and protein expression of fetal insulin receptor in breast cancer cell lines and tissues.

Breast cancer research and treatment·2026
Same author

Contribution of HMGB1 to keratinocyte inflammation in recessive dystrophic epidermolysis bullosa.

JID innovations : skin science from molecules to population health·2026
Same author

Distinct associations of pioneer factor Ascl1-E12a with nucleosomes drive changes in cell fate.

Molecular cell·2026
Same author

Impaired Lung BCAA Metabolism Promotes Ferroptosis and Resultant Pulmonary Arterial Hypertension-Associated Hepatopathy.

JACC. Basic to translational science·2026
Same author

Isotope Dilution NanoLC-MS/MS Quantitation of Methylglyoxal DNA-Protein Cross-Links: Formation and Repair in Human Cells.

Analytical chemistry·2026
Same author

Germline-targeting HIV immunogen induces cross-neutralizing antibodies in outbred macaques.

Immunity·2026

Related Experiment Video

Updated: Jun 27, 2025

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
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

338

Improved detection of DNA replication fork-associated proteins.

Rebecca S Rivard1, Ya-Chu Chang2, Ryan L Ragland1

  • 1Department of Cancer Biology and the Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Cell Reports
|May 4, 2024
PubMed
Summary

This study introduces iPOND2-DRIPPER, a method enhancing protein retrieval from replicating DNA. It reveals new insights into DNA replication fork stalling and the associated repair mechanisms.

Keywords:
CP: Molecular biologyDNA repairDNA replicationDRIPPERiPONDnuclear pore complexp97replication stressreplisomeubiquination

More Related Videos

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay
10:32

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

Published on: February 3, 2022

6.1K
Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles
13:09

Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles

Published on: March 3, 2023

3.1K

Related Experiment Videos

Last Updated: Jun 27, 2025

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
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

338
Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay
10:32

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

Published on: February 3, 2022

6.1K
Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles
13:09

Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles

Published on: March 3, 2023

3.1K

Area of Science:

  • Molecular Biology
  • Cellular Dynamics
  • Proteomics

Background:

  • Understanding DNA replication fork dynamics is crucial for cell cycle regulation.
  • Replication fork stalling can lead to genomic instability and disease.
  • Existing methods for studying replication-associated proteins have limitations in yield and quantification.

Purpose of the Study:

  • To develop and validate an enhanced method for identifying proteins at active DNA replication forks.
  • To investigate the proteomic changes associated with replication fork stalling.
  • To uncover the molecular players involved in DNA repair and cellular response to stalled replication.

Main Methods:

  • Utilized density-based replisome enrichment via isolating proteins on nascent DNA (iPOND2).
  • Combined iPOND2 with label-free quantitative mass spectrometry (iPOND2-DRIPPER) for enhanced protein analysis.
  • Enabled direct measurement of ubiquitination events on stalled replication machinery.

Main Results:

  • iPOND2-DRIPPER significantly increased replication factor yields (up to 300-fold) and protein quantification dynamic range.
  • Observed substantial recruitment of replication stress and DNA repair factors upon fork stalling.
  • Identified temporally controlled associations of stalled replisomes with nuclear pore complexes and cytoskeleton networks.

Conclusions:

  • iPOND2-DRIPPER is a powerful technique for studying the dynamics of DNA replication and protein interactions.
  • Stalled replication forks recruit diverse DNA repair factors, including ubiquitination pathways.
  • Replisome dynamics are linked to nuclear architecture components during replication stress.