title: RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment.
abstract: DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.
authors.affiliation: From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Curtis D
authors.identifier: http://orcid.org/0000-0002-7528-4058
authors.initials: CD
authors.lastname: Hodge
authors.affiliation: the Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada, the Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt.
authors.forename: Ismail H
authors.initials: IH
authors.lastname: Ismail
authors.affiliation: From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Ross A
authors.initials: RA
authors.lastname: Edwards
authors.affiliation: the Lawrence Berkeley National Laboratory, Berkeley, California 94704.
authors.forename: Greg L
authors.initials: GL
authors.lastname: Hura
authors.affiliation: From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Xiao
authors.affiliation: the Lawrence Berkeley National Laboratory, Berkeley, California 94704, the Department of Molecular and Cellular Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, and.
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.affiliation: the Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hendzel
authors.affiliation: From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, mark.glover@ualberta.ca.
authors.forename: J N Mark
authors.initials: JN
authors.lastname: Glover
mesh_terms: D000818:Animals; D019313:BRCA1 Protein; D018360:Crystallography, X-Ray; D053903:DNA Breaks, Double-Stranded; D051379:Mice; D020836:Protein Structure, Quaternary; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D019313:BRCA1 Protein; C000605931:Brca1 protein, mouse; D025521:Tumor Suppressor Proteins; C514982:Ube2n protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; C549817:Rnf8 protein, mouse; D044767:Ubiquitin-Protein Ligases
keywords: 53BP1; BRCA1; DNA damage response; E3 ubiquitin-protein ligase RNF8 (RNF8); RNF168; Ubc13; cell biology; ubiquitylation (ubiquitination); x-ray crystallography; x-ray scattering