title: Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation.
abstract: Pairing of a given E3 ubiquitin ligase with different E2s allows synthesis of ubiquitin conjugates of different topologies. While this phenomenon contributes to functional diversity, it remains largely unknown how a single E3 ubiquitin ligase recognizes multiple E2s, and whether identical structural requirements determine their respective interactions. The E3 ubiquitin ligase RNF8 that plays a critically important role in transducing DNA damage signals, interacts with E2s UBCH8 and UBC13, and catalyzes both K48- and K63-linked ubiquitin chains. Interestingly, we report here that a single-point mutation (I405A) on the RNF8 polypeptide uncouples its ability in catalyzing K48- and K63-linked ubiquitin chain formation. Accordingly, while RNF8 interacted with E2s UBCH8 and UBC13, its I405A mutation selectively disrupted its functional interaction with UBCH8, and impaired K48-based poly-ubiquitylation reactions. In contrast, RNF8 I405A preserved its interaction with UBC13, synthesized K63-linked ubiquitin chains, and assembled BRCA1 and 53BP1 at sites of DNA breaks. Together, our data suggest that RNF8 regulates K48- and K63-linked poly-ubiquitylation via differential RING-dependent interactions with its E2s UBCH8 and UBC13, respectively.
authors.affiliation: Genome Stability Research Laboratory, The University of Hong Kong, L1, Laboratory Block, 21 Sassoon Road, Hong Kong SAR.
authors.forename: Gabriel Tsz-Mei
authors.initials: GT
authors.lastname: Lok
authors.forename: Shirley M-H
authors.initials: SM
authors.lastname: Sy
authors.forename: Sui-Sui
authors.initials: SS
authors.lastname: Dong
authors.forename: Yick-Pang
authors.initials: YP
authors.lastname: Ching
authors.forename: Sai Wah
authors.initials: SW
authors.lastname: Tsao
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Thomson
authors.forename: Michael S Y
authors.initials: MS
authors.lastname: Huen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004249:DNA Damage; D008239:Lysine / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D055550:Protein Stability; D015398:Signal Transduction; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine