Related Experiment Video
Updated: Jun 11, 2025
07:55
Visualization of DNA Repair Proteins Interaction by Immunofluorescence
Published on: June 26, 2020
10.1K
A Site-Specific Click Chemistry Approach to Di-Ubiquitylate H1 Variants Reveals Position-Dependent Stimulation of the
Pauline Franz1, Charlotte M Delvaux de Fenffe1,2, Beat Fierz1
1Laboratory of Biophysical Chemistry of Macromolecules (LCBM), Institute of Chemical Sciences and Engineering (ISIC), EPFL (Ecole Polytechnique Fédérale de Lausanne), Station 6, 1015, Lausanne, Switzerland.
Angewandte Chemie (International Ed. in English)
|October 8, 2024
Summary
Researchers developed a click chemistry method to create poly-ubiquitylated histone H1, demonstrating its role in recruiting RNF168 for DNA repair and offering cancer therapy insights.
Area of Science:
- Biochemistry and Molecular Biology
- Genetics and Genomics
- Cancer Research
Background:
- Histone H2A ubiquitylation by RNF168 is crucial for DNA double-strand break (DSB) repair.
- RNF168's activity is thought to be regulated by K63-linked poly-ubiquitylation of histone H1, but direct evidence is lacking.
- Existing methods do not allow for specific poly-ubiquitylation of histone H1.
Purpose of the Study:
- To develop a method for site-specific poly-ubiquitylation of histone H1.
- To investigate the role of poly-ubiquitylated histone H1 in RNF168 recruitment and H2A ubiquitylation.
- To explore the potential of targeting RNF168 in cancer therapy.
Main Methods:
- Developed a versatile click chemistry approach for site-specific, stepwise protein conjugation.
- Synthesized histone H1 constructs with di-ubiquitin chains (H1^KxUb2) at four potential ubiquitylation sites.
- Integrated H1^KxUb2 variants into nucleosome arrays and tested their effect on RNF168 activity in vitro and in cells.
Main Results:
- The click chemistry method successfully generated H1^KxUb2 variants.
- H1^KxUb2 variants stimulated RNF168-mediated H2A ubiquitylation in a position-dependent manner, with H1^K17Ub2 being most effective.
- Di-ubiquitin binding to H1 was identified as the key factor for RNF168 recruitment.
- H1^K17Ub2 variants were successfully introduced into living cells, confirming RNF168 recruitment.
Conclusions:
- Poly-ubiquitylated histone H1 acts as a scaffold to recruit RNF168 to DSB sites, supporting the proposed mechanism.
- The developed click chemistry approach provides a powerful tool for studying RNF168 regulation.
- These findings have potential implications for developing novel cancer therapies targeting RNF168.
Related Concept Videos
01:38Nucleotide Excision Repair
3.5K
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...
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
01:02Long-patch Base Excision Repair
7.0K
Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
7.0K
02:53Conservative Site-specific Recombination and Phase Variation
5.9K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
5.9K
02:04Covalently Linked Protein Regulators
6.8K
Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein....
These groups modify specific amino acids in a protein....
6.8K
02:31Homologous Recombination
50.3K
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...
50.3K
02:37DNA 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