title: RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins.
abstract: DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.
authors.affiliation: Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Copenhagen, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Doil
authors.forename: Niels
authors.initials: N
authors.lastname: Mailand
authors.forename: Simon
authors.initials: S
authors.lastname: Bekker-Jensen
authors.forename: Patrice
authors.initials: P
authors.lastname: Menard
authors.forename: Dorthe Helena
authors.initials: DH
authors.lastname: Larsen
authors.forename: Rainer
authors.initials: R
authors.lastname: Pepperkok
authors.forename: Jan
authors.initials: J
authors.lastname: Ellenberg
authors.forename: Stephanie
authors.initials: S
authors.lastname: Panier
authors.forename: Daniel
authors.initials: D
authors.lastname: Durocher
authors.forename: Jiri
authors.initials: J
authors.lastname: Bartek
authors.forename: Jiri
authors.initials: J
authors.lastname: Lukas
authors.forename: Claudia
authors.initials: C
authors.lastname: Lukas
mesh_terms: D002460:Cell Line; D002875:Chromosomes / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006657:Histones / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D000071857:Tumor Suppressor p53-Binding Protein 1; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C486521:RNF8 protein, human; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D025801:Ubiquitin; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases