Related Experiment Video
Updated: Jun 25, 2026
In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
The Rap80-BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA
Genze Shao1, Dana R Lilli, Jeffrey Patterson-Fortin
1Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160, USA.
Abstract:
DNA double strand breaks (DSBs) initiate reversible cellular checkpoint and repair activities. Whereas many of the activating events at DSBs have recently been elucidated, the mechanisms used to terminate responses at these sites are largely undefined. Here we report a pathway required to reverse RNF8-Ubc13 dependent ubiquitination events on chromatin flanking DSBs. Inhibition of the Rap80-BRCC36 de-ubiquitinating enzyme complex partially restored DSB-associated ubiquitin levels following RNF8 knockdown or proteasome inhibition. Similarly, BRCC36 knockdown or expression of a BRCC36 de-ubiquitinating enzyme-inactive mutant rescued both 53BP1 recruitment to DSBs and ionizing radiation-induced gammaH2AX ubiquitination following RNF8 depletion, and mitigated ionizing radiation sensitivity resulting from RNF8 deficiency. Thus, concomitant and opposing RNF8-Ubc13 ubiquitin ligase and Rap80-BRCC36 ubiquitin hydrolysis activities are responsible for determining steady-state ubiquitin levels at DNA DSBs. These findings reveal a Rap80-BRCC36 dependent pathway that is required for appropriate DSB recruitment and repair responses.
Insights
This study identifies a crucial de-ubiquitinating enzyme complex, Rap80-BRCC36, that reverses ubiquitination at DNA double-strand breaks (DSBs). This pathway is essential for proper DNA repair and cellular response to DNA damage.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- DNA double-strand breaks (DSBs) trigger cellular responses involving checkpoint activation and repair.
- Mechanisms for terminating these responses at DSBs are not well understood.
Purpose of the Study:
- To elucidate the pathway responsible for reversing ubiquitination events at DSBs.
- To identify the enzymes involved in the termination of DNA damage response.
Main Methods:
- Investigated the role of the Rap80-BRCC36 de-ubiquitinating enzyme complex.
- Utilized RNF8 knockdown, proteasome inhibition, and BRCC36 knockdown/mutant expression.
- Assessed DSB-associated ubiquitin levels, 53BP1 recruitment, and gammaH2AX ubiquitination.
Main Results:
- Inhibition of Rap80-BRCC36 restored ubiquitin levels after RNF8 depletion or proteasome inhibition.
- BRCC36 manipulation rescued 53BP1 recruitment and gammaH2AX ubiquitination following RNF8 depletion.
- Mitigated ionizing radiation sensitivity in RNF8-deficient cells.
Conclusions:
- Concomitant RNF8-Ubc13 ligase and Rap80-BRCC36 hydrolysis activities regulate ubiquitin levels at DSBs.
- A Rap80-BRCC36 dependent pathway is vital for DSB recruitment and repair.
Related Concept Videos
01:38Nucleotide Excision Repair
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...
01:08Nucleotide Excision Repair
01:08Nucleotide Excision Repair
02:37Restarting Stalled Replication Forks
02:31Homologous Recombination
00:50Anaphase Promoting Complex