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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
Histone Ubiquitination by the DNA Damage Response Is Required for Efficient DNA Replication in Unperturbed S Phase
Jonas Andreas Schmid1, Matteo Berti1, Franziska Walser1
1Institute of Molecular Cancer Research, University of Zurich, Zurich 8057, Switzerland.
Abstract:
Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, γH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease.
Insights
The RNF168 pathway is crucial for DNA replication fork stability and restart. Its loss impairs DNA replication and fork progression, highlighting a new role in preventing genome instability.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Chromatin ubiquitination by RNF168 is vital for the DNA damage response (DDR).
- DDR deficiencies are linked to cancer, but the exact mechanisms, especially concerning DNA repair and replication, remain unclear.
Purpose of the Study:
- To investigate the role of the RNF168 pathway in DNA replication during unperturbed S phase.
- To determine if RNF168's function in DNA damage response extends to DNA replication fork dynamics.
Main Methods:
- Analysis of RNF168-deficient cells to assess DNA replication fork progression and stability.
- Investigating the role of MRE11 in reversed fork degradation.
- Assessing the requirement of H2A ubiquitination for RNF168's replication function.
Main Results:
- Loss of RNF168 significantly reduces replication fork progression and increases reversed fork accumulation, especially at repetitive DNA sequences.
- RNF168 is implicated in the protection and restart of reversed replication forks, with MRE11-dependent degradation playing a role in slow fork progression.
- RNF168's replication function is dependent on H2A ubiquitination, similar to other DDR factors like ATM, γH2A.X, RNF8, and 53BP1.
Conclusions:
- The RNF168 pathway plays a previously unrecognized role in maintaining DNA replication fork integrity.
- Reversed replication forks engage classical DNA damage response factors, suggesting a broader function in preventing genome instability and associated diseases.
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