Department of Molecular Genetics, University of Toronto, Toronto, Canada; Department of Biochemistry, University of Toronto, Toronto, Canada; Department of Chemistry, University of Toronto, Toronto, Canada
The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada; Laval University Cancer Research Center, Oncology Axis – Centre Hospitalier Universitaire de Québec Research Center – Université Laval, Hôtel-Dieu de Québec, Québec City, Canada
Predrag Kukic
Department of Chemistry, University of Cambridge, Cambridge, United Kingdom
Department of Molecular Genetics, University of Toronto, Toronto, Canada; Department of Biochemistry, University of Toronto, Toronto, Canada; Department of Chemistry, University of Toronto, Toronto, Canada
Stephanie Panier
Department of Molecular Genetics, University of Toronto, Toronto, Canada; The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada
Shili Duan
Structural Genomics Consortium, University of Toronto, Toronto, Canada; Princess Margret Cancer Centre, Toronto, Canada
Marella D Canny
Department of Medical Biophysics, University of Toronto, Toronto, Canada
Hugo van Ingen
Macromolecular Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
Cheryl H Arrowsmith
Structural Genomics Consortium, University of Toronto, Toronto, Canada; Princess Margret Cancer Centre, Toronto, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Canada
Department of Molecular Genetics, University of Toronto, Toronto, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Canada; Molecular Structure and Function Program, The Hospital for Sick Children Research Institute, Toronto, Canada
Department of Molecular Genetics, University of Toronto, Toronto, Canada; The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada
Department of Molecular Genetics, University of Toronto, Toronto, Canada; Department of Biochemistry, University of Toronto, Toronto, Canada; Department of Chemistry, University of Toronto, Toronto, Canada; Molecular Structure and Function Program, The Hospital for Sick Children Research Institute, Toronto, Canada
Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes.
