Abstract
RAD18, a RING-type ubiquitin ligase (E3) that plays an essential role in post-replication repair, possesses distinct domains named RING, UBZ, SAP and the RAD6-binding domain (R6BD) and forms a dimer. RAD6, an ubiquitin-conjugating enzyme (E2), stably associates with R6BD in the C-terminal portion. In this study, we established a method to distinguish between the two subunits of RAD18 by introduction of different tags, and analyzed mutant complexes. Our results, surprisingly, demonstrate that RAD6A and RAD18 form a ternary complex, RAD6A-(RAD18)(2) and the presence of only one R6BD in the two RAD18 subunits is sufficient for ternary complex formation and the ligase activity. Interestingly, ligase activity of a mutant dimer lacking both R6BDs is not restored even with large amounts of RAD6A added in solution, suggesting a requirement for precise juxtaposition via interaction with R6BD. We further show that mutations in both subunits of either RING or SAP, but not UBZ, strongly reduce ligase activity, although inactivation in only one of two subunits is without effect. These results suggest an asymmetric nature of the two RAD18 subunits in the complex.