RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.
We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does ... not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3Â ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells.
Mesh Terms:
Chromatin, DNA, DNA Damage, DNA Repair, DNA Replication, HeLa Cells, Homologous Recombination, Humans, Models, Molecular, Mutation, Protein Binding, Protein Subunits, Replication Protein A, Ubiquitin-Protein Ligases, Ubiquitination
Chromatin, DNA, DNA Damage, DNA Repair, DNA Replication, HeLa Cells, Homologous Recombination, Humans, Models, Molecular, Mutation, Protein Binding, Protein Subunits, Replication Protein A, Ubiquitin-Protein Ligases, Ubiquitination
Mol. Cell
Date: Oct. 15, 2015
PubMed ID: 26474068
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