title: RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.
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.
authors.affiliation: 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.
authors.forename: Andrew E H
authors.initials: AE
authors.lastname: Elia
authors.affiliation: 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.
authors.forename: David C
authors.initials: DC
authors.lastname: Wang
authors.affiliation: Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Willis
authors.affiliation: 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.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Boardman
authors.affiliation: Department of Genetics, Harvard Medical School; Division of Genetics, Brigham and Women's Hospital; Howard Hughes Medical Institute, Boston, MA 02115, USA.
authors.forename: Ildiko
authors.initials: I
authors.lastname: Hajdu
authors.affiliation: Department of Genetics, Harvard Medical School; Division of Genetics, Brigham and Women's Hospital; Howard Hughes Medical Institute, Boston, MA 02115, USA.
authors.forename: Richard O
authors.initials: RO
authors.lastname: Adeyemi
authors.affiliation: 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.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Lowry
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.affiliation: Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
authors.forename: Ralph
authors.initials: R
authors.lastname: Scully
authors.affiliation: 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.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Elledge
mesh_terms: D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D006367:HeLa Cells; D059765:Homologous Recombination; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D002843:Chromatin; D021122:Protein Subunits; D051716:Replication Protein A; D004247:DNA; C549038:RFWD3 protein, human; D044767:Ubiquitin-Protein Ligases