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Updated: May 25, 2026
Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair
Published on: May 24, 2017
The E3 ligase RNF8 regulates KU80 removal and NHEJ repair
1Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Abstract:
The ubiquitination cascade has a key role in the assembly of repair and signaling proteins at sites of double-strand DNA breaks. The E3 ubiquitin ligase RING finger protein 8 (RNF8) triggers the initial ubiquitination at double-strand DNA breaks, whereas sustained ubiquitination requires the downstream E3 ligase RING finger protein 168 (RNF168). It is not known whether RNF8 and RNF168 have discrete substrates and/or form different ubiquitin chains. Here we show that RNF168 acts with the ubiquitin-conjugating enzyme E2 13 (UBC13) and specifically synthesizes Lys63-linked chains, whereas RNF8 primarily forms Lys48-linked chains on chromatin, which promote substrate degradation. We also find that RNF8 regulates the abundance of the nonhomologous end-joining (NHEJ) repair protein KU80 at sites of DNA damage, and that RNF8 depletion results in prolonged retention of KU80 at damage sites and impaired nonhomologous end-joining repair. These findings reveal a distinct feature of RNF8 and indicate the involvement of the ubiquitination-mediated degradation pathway in DNA damage repair.
Insights
RING finger protein 8 (RNF8) and RING finger protein 168 (RNF168) play distinct roles in DNA double-strand break repair. RNF8 promotes KU80 degradation via Lys48-linked chains, essential for efficient repair.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Ubiquitination is crucial for DNA double-strand break (DSB) repair and signaling.
- RING finger protein 8 (RNF8) initiates ubiquitination at DSBs, while RING finger protein 168 (RNF168) sustains it.
Purpose of the Study:
- To investigate the distinct roles and substrate specificities of RNF8 and RNF168 in DNA damage response.
- To elucidate the types of ubiquitin chains synthesized by RNF8 and RNF168.
Main Methods:
- Analysis of ubiquitin chain synthesis by RNF8 and RNF168 in vitro and in vivo.
- Assessment of KU80 protein levels and localization at DNA damage sites.
- Evaluation of nonhomologous end-joining (NHEJ) repair efficiency following RNF8 depletion.
Main Results:
- RNF168 synthesizes Lys63-linked ubiquitin chains, while RNF8 primarily forms Lys48-linked chains.
- RNF8 regulates the abundance of KU80 at DNA damage sites.
- RNF8 depletion leads to prolonged KU80 retention and impaired NHEJ repair.
Conclusions:
- RNF8 and RNF168 exhibit distinct substrate specificities and ubiquitin chain formation.
- Ubiquitination-mediated degradation, regulated by RNF8, is a critical component of DNA damage repair pathways.
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