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RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly
Michael S Y Huen1, Robert Grant, Isaac Manke
1Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA.
Abstract:
DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 A. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.
Insights
RNF8 is crucial for the early DNA damage response, aiding in checkpoint protein accumulation and DNA repair. Cells lacking RNF8 show impaired cell cycle checkpoints and increased sensitivity to DNA damage.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA damage response (DDR) involves posttranslational modifications.
- Cell-cycle checkpoints, DNA repair, senescence, and apoptosis are regulated by DDR.
Purpose of the Study:
- To investigate the role of RNF8 in the early DNA damage response.
- To elucidate the molecular mechanisms by which RNF8 functions in DDR.
Main Methods:
- X-ray crystallography to determine the structure of the FHA domain of RNF8.
- Cellular assays to assess the impact of RNF8 depletion on DNA damage response pathways.
- Analysis of protein-protein interactions and ubiquitylation events.
Main Results:
- RNF8 facilitates the recruitment of BRCA1 and 53BP1 to damaged chromatin.
- RNF8 binds to MDC1 in a phospho-dependent manner via its FHA domain.
- RNF8 ubiquitinates H2AX and other substrates at DNA damage sites.
- RNF8-depleted cells exhibit a defective G2/M checkpoint and increased sensitivity to ionizing radiation (IR).
Conclusions:
- RNF8 is a novel DNA-damage-responsive protein.
- RNF8 integrates phosphorylation and ubiquitylation signaling in DDR.
- RNF8 plays a critical role in the cellular response to genotoxic stress.
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