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RNF8-dependent and RNF8-independent regulation of 53BP1 in response to DNA damage
1Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
Abstract:
The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G2/M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.
Insights
The proteasome regulates DNA damage response by controlling protein ubiquitylation and degradation. Proteasome inhibition delays ATM/ATR signaling and 53BP1 focus formation after UV damage, revealing distinct RNF8-dependent and independent pathways.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- DNA damage surveillance is crucial for genomic stability, involving ATM/ATR kinases and signaling cascades.
- Protein ubiquitylation and degradation are increasingly recognized as key regulators of DNA damage response (DDR).
Purpose of the Study:
- To investigate the role of the proteasome in DNA damage response pathways, specifically ATM/ATR signaling and checkpoint activation following UV irradiation.
- To elucidate the involvement of E3 ligase RNF8 and UBC13 in regulating 53BP1 localization and phosphorylation in response to different DNA damaging agents.
Main Methods:
- Utilized HeLa cells treated with proteasome inhibitor MG-132 to assess effects on UV-induced DNA damage signaling.
- Analyzed phosphorylation of ATM substrates and 53BP1 recruitment to DNA damage foci.
- Investigated the dependence of 53BP1 targeting and phosphorylation on RNF8 and UBC13 in response to UV and ionizing radiation.
- Assessed G2/M checkpoint activation following suppression of RNF8 or UBC13.
Main Results:
- Proteasome inhibition delayed UV-induced phosphorylation of ATM substrates and suppressed 53BP1 phosphorylation and focus formation.
- UV-induced recruitment of MDC1 and H2AX to DNA damage sites was unaffected by proteasome inhibition.
- RNF8 was critical for 53BP1 targeting and phosphorylation after ionizing radiation but only partially required for UV-induced 53BP1 regulation.
- Suppression of RNF8 or UBC13 caused subtle defects in UV-induced G2/M checkpoint activation.
Conclusions:
- The proteasome plays a significant role in regulating ATM/ATR signaling and 53BP1 dynamics during the DNA damage response to UV light.
- RNF8-dependent ubiquitylation pathways are essential for 53BP1 regulation following ionizing radiation.
- RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light, indicating distinct regulatory mechanisms for different DNA damage types.
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