title: Molecular insights into the function of RING finger (RNF)-containing proteins hRNF8 and hRNF168 in Ubc13/Mms2-dependent ubiquitylation.
abstract: The repair of DNA double strand breaks by homologous recombination relies on the unique topology of the chains formed by Lys-63 ubiquitylation of chromatin to recruit repair factors such as breast cancer 1 (BRCA1) to sites of DNA damage. The human RING finger (RNF) E3 ubiquitin ligases, RNF8 and RNF168, with the E2 ubiquitin-conjugating complex Ubc13/Mms2, perform the majority of Lys-63 ubiquitylation in homologous recombination. Here, we show that RNF8 dimerizes and binds to Ubc13/Mms2, thereby stimulating formation of Lys-63 ubiquitin chains, whereas the related RNF168 RING domain is a monomer and does not catalyze Lys-63 polyubiquitylation. The crystal structure of the RNF8/Ubc13/Mms2 ternary complex reveals the structural basis for the interaction between Ubc13 and the RNF8 RING and that an extended RNF8 coiled-coil is responsible for its dimerization. Mutations that disrupt the RNF8/Ubc13 binding surfaces, or that truncate the RNF8 coiled-coil, reduce RNF8-catalyzed ubiquitylation. These findings support the hypothesis that RNF8 is responsible for the initiation of Lys-63-linked ubiquitylation in the DNA damage response, which is subsequently amplified by RNF168.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Campbell
authors.forename: Ross A
authors.initials: RA
authors.lastname: Edwards
authors.forename: Charles C Y
authors.initials: CC
authors.lastname: Leung
authors.forename: Dante
authors.initials: D
authors.lastname: Neculai
authors.forename: Curtis D
authors.initials: CD
authors.lastname: Hodge
authors.forename: Sirano
authors.initials: S
authors.lastname: Dhe-Paganon
authors.forename: J N Mark
authors.initials: JN
authors.lastname: Glover
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; C486521:RNF8 protein, human; D011994:Recombinant Proteins; C471607:UBE2N protein, human; C114715:UBE2V2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D008239:Lysine