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.
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Abbreviations
- DSBs:
-
double-strand breaks
- HR:
-
homologous recombination
- NHEJ:
-
non-homologous end joining
- IR:
-
ionizing radiation
- UV:
-
ultraviolet
- IRIF:
-
ionizing radiation-induced foci
- DDR:
-
DNA damage response
- STUbl:
-
SUMO-targeted ubiquitin ligase
- SA:
-
splice acceptor
- PCR:
-
polymerase chain reaction
- MEFs:
-
mouse embryonic fibroblasts
- RT-qPCR:
-
reverse transcription quantitative PCR
- ROS:
-
reactive oxygen species
- PFGE:
-
pulsed-field gel electrophoresis
- IHC:
-
immunohistochemistry
- RING:
-
really interesting new gene
- H&E:
-
hematoxylin and eosin
- (E)GFP:
-
(enhanced) green fluorescent protein
- Hypo:
-
hypomorphic
- SIM:
-
SUMO interaction motif
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Acknowledgements
We thank Odessa Van Goethem for excellent technical assistance and Martijn Luijsterburg for help with the UV-A laser micro-irradiation experiments. This work was supported by the Belgian federation against cancer (BFK), the Association for International Cancer Research (AICR), the Flemish Organisation for Scientific Research (FWO ZKC2592-00_WO1), and by the Netherlands Organisation for Scientific Research (NWO).
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Vyas, R., Kumar, R., Clermont, F. et al. RNF4 is required for DNA double-strand break repair in vivo. Cell Death Differ 20, 490–502 (2013). https://doi.org/10.1038/cdd.2012.145
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DOI: https://doi.org/10.1038/cdd.2012.145
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