Related Experiment Video

Updated: Nov 21, 2025

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
08:31

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Published on: June 8, 2018

9.4K

Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA

Kyosuke Nakamura1, Georg Kustatscher2, Constance Alabert3

  • 1The Novo Nordisk Center for Protein Research (CPR), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark; Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.

Molecular Cell
|January 15, 2021
PubMed

Insights

This study reveals how cells repair DNA double-strand breaks (DSBs) during replication using nascent chromatin capture proteomics. ATM signaling plays a key role in DNA repair pathway choice and fork proteome regulation.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Cellular DNA repair mechanisms are crucial for accurate DNA replication and preventing genome instability.
  • Defects in DNA repair can lead to cancer and present therapeutic targets.
  • Replication-associated DNA double-strand breaks (DSBs) are a significant challenge during DNA replication.

Purpose of the Study:

  • To characterize the proteome of stalled and broken replication forks using nascent chromatin capture (NCC) proteomics.
  • To investigate the role of ATM signaling in the DNA repair response to TOP1 inhibitor-induced DSBs.

Main Methods:

  • Nascent chromatin capture (NCC) proteomics was employed to analyze the protein environment at replication forks.
  • The study utilized topoisomerase 1 (TOP1) inhibitors to induce replication-associated DSBs.
  • ATM inhibition was used to probe the downstream signaling effects on the fork proteome.

Main Results:

  • NCC proteomics revealed significant alterations in the fork proteome, including chromatin and nuclear membrane interactions.
  • Three distinct classes of repair factors were identified based on their enrichment at broken and/or stalled forks.
  • ATM inhibition altered the broken fork proteome, promoting DNA end resection and PLK1 recruitment while suppressing canonical DSB ubiquitination by RNF168 and BRCA1-A.

Conclusions:

  • This study provides a comprehensive proteomic map of replication fork repair.
  • The findings elucidate the critical role of ATM signaling in orchestrating homologous recombination repair of replication-associated DSBs.
  • The results offer a new framework for understanding DNA repair dynamics at stalled replication forks.
Keywords:
ATMBRCA1-ACamptothecinNDRG3NHEJPLK1UBAP2homologous recombinationnascent chromatin capturereplication stress

More Related Videos

Visualization of DNA Repair Proteins Interaction by Immunofluorescence07:55

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Published on: June 26, 2020

10.7K
Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage10:44

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Published on: January 31, 2018

10.5K

Related Concept Videos

DNA Damage can Stall the Cell Cycle02:37

DNA Damage can Stall the Cell Cycle

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...
9.7K
DNA Damage Can Stall the Cell Cycle02:37

DNA Damage Can Stall the Cell Cycle

2.9K
Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

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,...
6.1K
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
59.3K
Fixing Double-strand Breaks02:04

Fixing Double-strand Breaks

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
13.5K
Fixing Double-strand Breaks02:04

Fixing Double-strand Breaks

3.9K