Abstract Background and Objectives: The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. A longstanding question in this field is how a single lysine residue in the nucleosome is singled out for ubiquitin modification and why only one ubiquitin is added rather than a chain of ubiquitins. To better understand how Bmi1/Ring1b controls monoubiquitination of H2A, we structurally characterized the E2 and nucleosome interaction sites on Bmi1/Ring1b. Methods: X-ray crystallography was used to determine the structure of a complex between Bmi1/Ring1b and UbcH5c, its cognate E2 enzyme. Fluorescence-polarization assays were used to measure the binding of Bmi1/Ring1b to a synthetic DNA duplex. Site-directed mutagenesis was used to assess the importance of residues at the potential Bmi1/Ring1b-DNA interface, and in vitro ubiquitin ligase assays were used to measure the catalytic activity of mutant complexes. Molecular modeling studies were performed using HADDOCK v2.0. Results: We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Through site-directed mutagenesis we have been able to uncouple the E2 binding and the DNA binding activities of Bmi1/Ring1b, indicating that Bmi1/Ring1b uses distinct binding surfaces to recognize E2 and the nucleosomal substrate. Computational modeling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific mono-ubiquitination of H2A. Conclusions: Our data show that the direct interaction of the RING domains with nucleosomal DNA is crucial for the ubiquitin ligase activity of Bmi1/Ring1b. To our knowledge, this is the first example of a RING-domain E3 ligase binding directly to its substrate via the RING domain. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A160.