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.
Subject(s)
DNA Repair , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing , Alleles , Animals , BRCA1 Protein/metabolism , Cell Cycle Proteins , Cell Line , DNA Breaks, Double-Stranded , Genotype , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Transgenic , Nuclear Proteins/genetics , Rad51 Recombinase/metabolism , Radiation, Ionizing , Sumoylation , Transcription Factors/genetics , Ubiquitin-Protein Ligases , UbiquitinationABSTRACT
Post-translational modification of proteins by SUMOs (small ubiquitin-related modifiers) plays an important role in a wide variety of biological processes. The mammalian SUMO family includes three members, SUMO-1, SUMO-2 and SUMO-3. While target proteins are predominantly conjugated to monomeric SUMO, all three SUMO family members are able to multimerize in vitro. In cells, SUMOs have the potential to multimerize via internal consensus sites for SUMOylation that are present in SUMO-2 and SUMO-3. A SUMO-binding motif in Ubc9 (ubiquitin-conjugating enzyme 9) contributes to SUMO chain formation in vitro and SUMO E3 ligases further enhance SUMO polymerization. SUMO chain formation is reversible; SUMO polymers are disassembled by SUMO proteases both in vitro and in vivo. Despite recent progress, the functional relevance of SUMO polymerization is still unclear and little is known about the identity of the endogenous target proteins that are conjugated to SUMO polymers.
