Symmetry and Asymmetry of the RING–RING Dimer of Rad18 Anding Huang 1 †, Richard G. Hibbert 2 †, Rob N. de Jong 1 , Devashish Das 1 , Titia K. Sixma 2 ⁎ and Rolf Boelens 1 ⁎ 1 Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands 2 Division of Biochemistry and Center for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Received 13 January 2011; received in revised form 14 April 2011; accepted 20 April 2011 Available online 27 April 2011 Edited by K. Morikawa Keywords: ubiquitination; Rad18; Rad6; NMR; X-ray crystallography The human ubiquitin-conjugating enzyme Rad6 (E2), with ubiquitin ligase enzyme Rad18 (RING E3), monoubiquitinates proliferating cell nuclear antigen at stalled replication forks in DNA translesion synthesis. Here, we determine the structure of the homodimeric Rad18 RING domains by X-ray crystallography and classify it to RING–RING dimers that dimerize through helices adjacent to the RING domains and through the canonical RING domains. Using NMR spectroscopy and site-directed mutagenesis, we demonstrate that the Rad6b binding site, for the Rad18 RING domain, strongly resembles that of other E2/E3 RING/U-box complexes. We show that the homodimeric Rad18 RING domain can recruit two Rad6b E2 enzymes, whereas the full-length Rad18 homodimer binds only to a single Rad6b molecule. Such asymmetry is a common feature of RING–RING heterodimers and has been observed for the CHIP U-box homodimer. We propose that asymmetry may be a common feature of dimeric RING E3 ligases. © 2011 Elsevier Ltd. All rights reserved. Introduction Protein modification by ubiquitin or ubiquitin- like proteins through the E1-E2-E3 enzyme cas- cade has been known not only for its role in proteolytic degradation but also for modulation of protein function, 1 as well as in regulating DNA replication and DNA repair. 2,3 Ubiquitin modifi- cation of proliferating cell nuclear antigen (PCNA), a DNA polymerase sliding clamp that encircles DNA as a homotrimeric protein complex, has emerged as an important regulatory mecha- nism to bypass DNA lesions during replication and maintain gene stability. 4–6 This DNA lesion bypass mechanism is known as DNA damage tolerance and consists of mainly two pathways: the translesion synthesis (TLS) and the template switch (error-free) according to their potential to cause mutations. 7 *Corresponding authors. E-mail addresses: t.sixma@nki.nl; r.boelens@uu.nl. † A.H. and R.G.H. contributed equally to this work. Present addresses: R. N. de Jong, Genmab B.V., Yalelaan 60, 3584 CM Utrecht, The Netherlands; D. Das, Department of Cell Biology and Genetics, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands. Abbreviations used: PCNA, proliferating cell nuclear antigen; TLS, translesion synthesis; R6BD, Rad6 binding domain; MALLS, multi-angle laser light scattering; PDB, Protein Data Bank; CSP, chemical shift perturbation; ITC, isothermal titration calorimetry; TCEP, tris(2- carboxyethyl)phosphine; PEG, polyethylene glycol; MME, monomethyl ether. doi:10.1016/j.jmb.2011.04.051 J. Mol. Biol. (2011) 410, 424–435 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.