Ubiquitination of target proteins is accomplished by isopeptide bond formation between the carboxy group of the C-terminal glycine (Gly) residue of ubiquitin (Ub) and the epsilon-amino group of lysine (Lys) on the target proteins. The formation of an isopeptide bond between Ubs that gives rise to a poly-Ub chain on the target proteins and the types of poly-Ub chains formed depend on which of the seven Lys residues or N-terminal methionine (Met) residue on Ub is used for chain elongation. To understand the linkage specificity mechanism of Ub chains on E3, the previous study established an assay to monitor the formation of a free diubiquitin chain (Ub(2) chain synthesis assay) by HECT type E3 figase. In this study, we investigated Ub(2) chain specificity using E6AP HECT domain. We here demonstrate the importance of the N-terminal domain of full length E6AP for Ub(2) chain specificity. (C) 2017 Elsevier Inc. All rights reserved.