Molecular Insights into the Function of RING Finger (RNF)-containing Proteins hRNF8 and hRNF168 in Ubc13/Mms2-dependent Ubiquitylation

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. Background: RNF8 and RNF168 are essential RING-E3 ubiquitin ligases that catalyze the formation of Lys-63 ubiquitin chains in the DNA damage response (DDR). Results: We solved the crystal structures and probed the activity of RNF8 and RNF168 in vitro. Conclusion: RNF168 likely acts downstream of RNF8 given its deficient activity. Significance: Our data provide structural and catalytic insight into the relative activities of RNF8 and RNF168.