2. Academic Validation
  3. RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks

  • Mol Cell. 2015 Oct 15;60(2):280-93. doi: 10.1016/j.molcel.2015.09.011.
Andrew E H Elia 1,  David C Wang 1,  Nicholas A Willis 2,  Alexander P Boardman 1,  Ildiko Hajdu 3,  Richard O Adeyemi 3,  Elizabeth Lowry 1,  Steven P Gygi 4,  Ralph Scully 2,  Stephen J Elledge 5
Affiliations

Affiliations

  • 1 Department of Genetics, Harvard Medical School; Division of Genetics, Brigham and Women's Hospital; Howard Hughes Medical Institute, Boston, MA 02115, USA; Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA 02114, USA.
  • 2 Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
  • 3 Department of Genetics, Harvard Medical School; Division of Genetics, Brigham and Women's Hospital; Howard Hughes Medical Institute, Boston, MA 02115, USA.
  • 4 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
  • 5 Department of Genetics, Harvard Medical School; Division of Genetics, Brigham and Women's Hospital; Howard Hughes Medical Institute, Boston, MA 02115, USA. Electronic address: selledge@genetics.med.harvard.edu.
PMID: 26474068 DOI: 10.1016/j.molcel.2015.09.011
Abstract

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 E3 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.

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