Related Experiment Video

Updated: Jul 5, 2025

Visualization of DNA Repair Proteins Interaction by Immunofluorescence
07:55

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Published on: June 26, 2020

10.2K

Ku80 is indispensable for repairing DNA double-strand breaks at highly methylated sites in human HCT116 cells

Mengtan Xing1, Yanhong Xiong1, Yong Zhang1

  • 1Institute for Regenerative Medicine, Department of Neurosurgery, Shanghai East Hospital, Shanghai Key Laboratory of Signaling and Disease Research, Frontier Science Center for Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

DNA Repair
|January 14, 2024
PubMed

Insights

Ku80 knockout human cells accumulate DNA double-strand breaks (DSBs) at methylated DNA regions. This study highlights Ku80 knockout cells as a model for studying structural variants and alternative-end joining repair.

Area of Science:

  • Genomics
  • Cell Biology
  • Molecular Biology

Background:

  • DNA double-strand breaks (DSBs) are critical DNA lesions that can lead to cell death.
  • Accumulating DSBs for genomic analysis is challenging.
  • Understanding DSB distribution and chromatin impact is vital.

Purpose of the Study:

  • To investigate DSB accumulation and genomic distribution in Ku80 knockout human cells.
  • To analyze the impact of DSBs on chromatin structure and identify structural variants.
  • To establish a model for studying alternative-end joining (Alt-EJ) repair.

Main Methods:

  • CRISPR-Cas9 technology to generate Ku80 knockout (Ku80-/-) human HCT116 cells.
  • Cell cycle arrest in G1 phase to accumulate DSBs.
  • End-seq and Nanopore sequencing for DNA analysis and structural variant identification.

Main Results:

  • DSB hotspots were identified in highly methylated DNA regions in Ku80-/- cells.
  • A significant increase in chromosome structural variants (SVs) was observed in Ku80-/- cells.
  • High efficiency of Ku80 knockout in HCT116 cells was confirmed.

Conclusions:

  • Ku80 knockout human cells provide a robust model for characterizing SVs within the 3D chromatin context.
  • This model facilitates the study of the alternative-end joining (Alt-EJ) DSB repair pathway.
  • Methylated DNA regions are associated with DSB hotspots in the absence of Ku80.
Keywords:
CRIPSRChromatinEND-seqNanoporeStructural variants

More Related Videos

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins10:24

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Published on: September 28, 2012

14.1K
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

10.3K

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...
9.2K
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...
50.6K
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...
12.6K
Nucleotide Excision Repair01:38

Nucleotide Excision Repair

DNA Distortion and Damage
Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...
3.5K
Mismatch Repair01:20

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
4.9K
Base Excision Repair01:54

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
22.4K