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Updated: May 16, 2026
Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells
Published on: September 8, 2010
RNF4 is required for DNA double-strand break repair in vivo
1Center for the Biology of Disease, Laboratory for Molecular Cancer Biology, VIB, Leuven, Belgium.
Abstract:
Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events.
Insights
Rnf4, a ubiquitin ligase, is crucial for DNA double-strand break (DSB) repair by integrating SUMOylation and ubiquitylation. Rnf4 deficiency impairs DNA repair, causing sensitivity to genotoxic stress and reproductive issues.
Area of Science:
- Molecular Biology
- Cellular Biology
- Genetics
Background:
- Unrepaired DNA double-strand breaks (DSBs) lead to genetic instability, cancer, or cell death.
- Protein SUMOylation and ubiquitylation are vital for dynamic protein complex assembly at DNA damage sites.
Purpose of the Study:
- To investigate the role of Rnf4, an E3 ubiquitin ligase, in DNA double-strand break (DSB) repair.
- To elucidate how SUMOylation influences protein ubiquitylation at DSBs and the in vivo function of Rnf4 in the DNA damage response (DDR).
Main Methods:
- Generated an Rnf4 allelic series in mice to study its role in vivo.
- Assessed DNA damage, signaling, and sensitivity to genotoxic stress in Rnf4-deficient cells and mice.
- Investigated the molecular mechanism by which Rnf4 functions in DSB repair, including its targeting of SUMOylated proteins and role in Rad51 loading.
Main Results:
- Rnf4 accumulates at DSB repair foci and is essential for both homologous recombination (HR) and non-homologous end joining (NHEJ) repair.
- Rnf4 deficiency results in persistent DNA damage and signaling, increased sensitivity to genotoxic stress, and age-dependent spermatogenesis impairment.
- Rnf4 targets SUMOylated MDC1 and BRCA1, and is required for Rad51 loading at DNA damage sites.
Conclusions:
- Rnf4 is a critical component of the DNA damage response (DDR) in vivo.
- Rnf4 integrates SUMOylation and ubiquitylation events to control protein localization at DNA damage sites, thereby facilitating efficient DNA repair.
- Rnf4 plays a significant role in maintaining genomic stability and reproductive health.
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