RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

• Published in: The Journal of biological chemistry 2016-04, Vol.291 (18), p.9396-9410
• Main Authors: Hodge, Curtis D., Ismail, Ismail H., Edwards, Ross A., Hura, Greg L., Xiao, Andrew T., Tainer, John A., Hendzel, Michael J., Glover, J. N. Mark
• Format: Article
• Language: English
• Subjects: 53BP1; 60 APPLIED LIFE SCIENCES; Animals; BASIC BIOLOGICAL SCIENCES; BRCA1; BRCA1 Protein; cell biology; Crystallography, X-Ray; DNA Breaks, Double-Stranded; DNA damage response; E3 ubiquitin-protein ligase RNF8; E3 ubiquitin-protein ligase RNF8 (RNF8); Mice; Protein Structure and Folding; Protein Structure, Quaternary; RNF168; Signal Transduction; Tumor Suppressor Proteins - chemistry; Tumor Suppressor Proteins - genetics; Tumor Suppressor Proteins - metabolism; Ubc13; Ubiquitin-Conjugating Enzymes - chemistry; Ubiquitin-Conjugating Enzymes - genetics; Ubiquitin-Conjugating Enzymes - metabolism; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; ubiquitylation (ubiquitination); x-ray crystallography; x-ray scattering
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.715698

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.